Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles

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(A) «Cuarentena + relajamiento»: modelo ajustado en tres secciones: 09/03/2020-19/03/2020 (una población, C=20), 20/03/2020-10/05/2020 (dos subpoblaciones: 75% con , 25% con ), 11/05/2020-22/06/2020 (dos subpoblaciones: 75% con L=2, 34, 25% con H=2, 85). «Cuarentena»: modelo ajustado en dos secciones: 09/03/2020-19/03/2020 (una población, ), 20/03/2020-10/05/2020 (dos subpoblaciones: 75% con L=0, 5, 25% con ), se extrapolaron las nuevas infecciones para el período del 11/05/2020-22/06/2020 suponiendo que la población mantenía los parámetros del segundo ajuste. (B) Se presentan los mismos modelos ajustados que en A, junto con el modelo «Sin cuarentena» (modelo ajustado para el período 09/03/2020-19/03/2020 (una población, ) y extrapolado para el período 20/03/2020-22/06/2020 suponiendo que la población mantenía los parámetros de este ajuste. Parámetros fijos: , y ). El eje vertical se muestra en escala logarítmica para facilitar la comparación." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Nicolás Morando, Mauricio Sanfilippo, Francisco Herrero, Matías Iturburu, Ariel Torti, Daniel Gutson, María A. Pando, Roberto Daniel Rabinovich" "autores" => array:8 [ 0 => array:2 [ "nombre" => "Nicolás" "apellidos" => "Morando" ] 1 => array:2 [ "nombre" => "Mauricio" "apellidos" => "Sanfilippo" ] 2 => array:2 [ "nombre" => "Francisco" "apellidos" => "Herrero" ] 3 => array:2 [ "nombre" => "Matías" "apellidos" => "Iturburu" ] 4 => array:2 [ "nombre" => "Ariel" "apellidos" => "Torti" ] 5 => array:2 [ "nombre" => "Daniel" "apellidos" => "Gutson" ] 6 => array:2 [ "nombre" => "María A." "apellidos" => "Pando" ] 7 => array:2 [ "nombre" => "Roberto Daniel" "apellidos" => "Rabinovich" ] ] ] ] "resumen" => array:1 [ 0 => array:3 [ "titulo" => "Highlights" "clase" => "author-highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•Los modelos matemáticos permiten evaluar la evolución de la pandemia por SARS-CoV-2.</li><li class="elsevierStyleListItem" id="lsti0010">•El modelo desarrollado contempla subpoblaciones con distintas tasas de contactos.</li><li class="elsevierStyleListItem" id="lsti0015">•Las predicciones del SEIR-HL son más optimistas que las del SEIR.</li><li class="elsevierStyleListItem" id="lsti0020">•El cálculo modificado de R0 permite comparar distintas intervenciones.</li><li class="elsevierStyleListItem" id="lsti0025">•El modelo propuesto permite una mejor toma de decisiones en salud pública.</li></ul>" ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S032575412100078X?idApp=UINPBA00004N" "url" => "/03257541/0000005400000002/v2_202206160255/S032575412100078X/v2_202206160255/es/main.assets" ] "itemAnterior" => array:18 [ "pii" => "S0325754122000505" "issn" => "03257541" "doi" => "10.1016/j.ram.2022.05.007" "estado" => "S300" "fechaPublicacion" => "2022-04-01" "aid" => "501" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "sco" "cita" => "Rev Argent Microbiol. 2022;54:71-3" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:1 [ "total" => 0 ] "es" => array:10 [ "idiomaDefecto" => true "cabecera" => "Editorial" "titulo" => "Microbiología, bioeconomía y objetivos de desarrollo sostenible" "tienePdf" => "es" "tieneTextoCompleto" => "es" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "71" "paginaFinal" => "73" ] ] "titulosAlternativos" => array:1 [ "en" => array:1 [ "titulo" => "Microbiology, Bioeconomy and Sustainable Development Goals" ] ] "contieneTextoCompleto" => array:1 [ "es" => true ] "contienePdf" => array:1 [ "es" => true ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Inés Eugenia García de Salamone" "autores" => array:1 [ 0 => array:2 [ "nombre" => "Inés Eugenia" "apellidos" => "García de Salamone" ] ] ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754122000505?idApp=UINPBA00004N" "url" => "/03257541/0000005400000002/v2_202206160255/S0325754122000505/v2_202206160255/es/main.assets" ] "en" => array:21 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "74" "paginaFinal" => "80" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Pablo Farace, Silvio Cravero, Catalina Taibo, Julián Diodati, Claudia Morsella, Fernando Paolicchi, Julia Sabio y García, Andrea Gioffré" "autores" => array:8 [ 0 => array:3 [ "nombre" => "Pablo" "apellidos" => "Farace" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "Silvio" "apellidos" => "Cravero" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "Catalina" "apellidos" => "Taibo" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 3 => array:3 [ "nombre" => "Julián" "apellidos" => "Diodati" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 4 => array:3 [ "nombre" => "Claudia" "apellidos" => "Morsella" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 5 => array:3 [ "nombre" => "Fernando" "apellidos" => "Paolicchi" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 6 => array:3 [ "nombre" => "Julia" "apellidos" => "Sabio y García" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 7 => array:4 [ "nombre" => "Andrea" "apellidos" => "Gioffré" "email" => array:1 [ 0 => "gioffre.andrea@inta.gob.ar" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Instituto de Agrobiotecnología y Biología Molecular, INTA-CONICET, CICVyA-CNIA, Hurlingham, Buenos Aires, Argentina" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Laboratorio Integral de Microscopía, CICVyA, INTA Hurlingham, Buenos Aires, Argentina" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Laboratorio de Bacteriología-Departamento de Sanidad Animal EEA-INTA Balcarce, Buenos Aires, Argentina" "etiqueta" => "c" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Campylobacter fetus secreta vesículas de membrana externa inmunorreactivas que conservan la capa S" ] ] "resumenGrafico" => array:2 [ "original" => 1 "multimedia" => array:5 [ "identificador" => "fig0025" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => false "mostrarDisplay" => true "figura" => array:1 [ 0 => array:4 [ "imagen" => "fx1.jpeg" "Alto" => 515 "Ancho" => 1333 "Tamanyo" => 53066 ] ] ] ] "textoCompleto" => "IntroductionThe biogenesis and release of outer membrane vesicles (OMVs) is a common feature of several gram-negative bacteria. Through the release of OMVs and the subsequent fusion of membranes, different bacterial components could be delivered and incorporated into the host cells.Since their first description in the literature, OMVs have been associated to relevant functions related to host-pathogen (and bacteria–bacteria) interaction, such as delivery of virulence factors and immune modulators, disruption of host tissue, gene and RNA transfer among others<a class="elsevierStyleCrossRefs" href="#bib0235">14,25</a>.Campylobacter are microaerophilic, motile, non-fermentative gram-negative organisms. Most species of Campylobacter can cause disease in both humans and animals. Campylobacter fetus comprises two subspecies associated to mammal hosts (Campylobacter fetus fetus and Campylobacter fetus venerealis). In animals, they have been associated to bovine infertility and bovine/ovine abortion<a class="elsevierStyleCrossRef" href="#bib0270">21</a>. C. fetus fetus has been isolated from blood, abscess fluid, bone marrow, cerebrospinal fluid, joint prostheses or implanted catheters and biopsies or stool from humans with different clinical conditions such as fever, colitis, venous thrombosis, erysipelas, meningitis, pleuritic chest pain, lethargy, endocarditis, photophobia and gastrointestinal problems<a class="elsevierStyleCrossRefs" href="#bib0205">8,31</a>.Campylobacter spp. have an outermost surface component, which is called the surface layer (S-layer), which consists of a crystalline array of proteinaceous subunits (SLP or surface layer proteins)<a class="elsevierStyleCrossRef" href="#bib0295">26</a> encoded by a diverse number of homologous genes in each strain<a class="elsevierStyleCrossRef" href="#bib0310">29</a>. In C. fetus, SLPs attach to either type A or B lipopolysaccharides<a class="elsevierStyleCrossRef" href="#bib0305">28</a>. This bidimensional structure, which is external to the cell wall, fulfils a variety of biological functions and roles<a class="elsevierStyleCrossRef" href="#bib0210">9</a>. The S-layer has been characterized as a virulence factor that mediates the evasion of the immune response in C. fetus<a class="elsevierStyleCrossRefs" href="#bib0180">3,4</a>.Different virulence strategies have been widely described in the emblematic member of the genus: C. jejuni. One of the most innovative strategies is the delivery of the biologically active toxin CDT through OMVs<a class="elsevierStyleCrossRef" href="#bib0245">16</a>. While C. jejuni virulence has been widely studied, the description of C. fetus-host interphase has been delayed. In addition, OMVs have immunological properties that are suitable for vaccine strategies<a class="elsevierStyleCrossRef" href="#bib0300">27</a>. Interestingly, a Neisseria meningitidis OMV-based vaccine, the first licensed vaccine based on these nanoparticles, has demonstrated its usefulness in human health<a class="elsevierStyleCrossRefs" href="#bib0225">12,23</a>.In Argentina, reproductive diseases impair livestock production and C. fetus is among the main bacterial pathogens isolated from abortions in one of the most productive regions of the country<a class="elsevierStyleCrossRef" href="#bib0265">20</a>. C. fetus is the etiological agent of the venereal disease called bovine genital campylobacteriosis. Vaccination is not compulsive; however, it is often applied when positive animals are detected in the herd. The limited available data around bacterin-based commercial vaccines suggest that they must be improved<a class="elsevierStyleCrossRefs" href="#bib0190">5,7</a>. The use of novel adjuvants could improve the accuracy of current vaccines.The aim of this study was to evaluate the biogenesis of OMVs in C. fetus, to characterize them morphometrically and to assay their immunoreactivity.Material and methodsBacterial strains. Campylobacter fetus strains from the culture collection of the Bacteriology laboratory EEA-INTA Balcarce were used in this study. Three well-characterized C. fetus field strains obtained from tissue samples from bovine aborted fetuses were analyzed in the study: Campylobacterfetus venerealis biovar intermedius 06-341 (Buenos Aires), Campylobacterfetus fetus 13-344 (Buenos Aires), C. fetus fetus 08-421 (Santa Fe). The type strain C. fetus venerealis NCTC 10354 isolated from bovine vaginal mucus was added in the analyses. We have previously typed the strains following standard biochemical procedures<a class="elsevierStyleCrossRef" href="#bib0270">21</a>, whereas other studies have characterized them at molecular and genomic levels as well<a class="elsevierStyleCrossRefs" href="#bib0215">10,30</a>.Growth conditions and isolation of OMVs. OMVs were obtained by differential centrifugation from bacterial culture supernatants essentially as previously described by Elmi and co-workers for C. jejuni with some modifications<a class="elsevierStyleCrossRef" href="#bib0195">6</a>. Skirrow agar plates (Oxoid) were used for the recovery of the C.fetus strains. Strains were grown at 37°C for 72h (5% O2, 10% CO2 and 85% N2) and then the colonies were inoculated into 10ml Brucella broth (Oxoid) (starter culture). The cultures were grown on an orbital shaker (100rpm) under microaerophilic conditions at 37°C for 72h. Then, the starter culture was transferred to a flask containing 500ml Brucella broth. Because C. fetus is a slow growing bacterium, long-term cultures were grown to obtain the appropriate optical density (OD 0.3–0.4 reached after 72h–96h of culture). The bacterial culture was centrifuged at 10000g for 30min and the resulting supernatants were filtered through a 0.22μm membrane (Millipore) to remove cells and debris. Then, ultracentrifugation was carried out at 150000×g for 3h at 4°C using a 45Ti rotor (Beckman instruments) and the pellet containing OMVs was resuspended with a minimal volume of sterile phosphate-buffered saline pH 7.4 (PBS). Two further washing steps were performed with PBS before carrying out the immunoassays.Transmission electron microscopy (TEM) and morphometry. The OMV fraction was fixed with one volume of 4% paraformaldehyde, mounted on grids and negatively stained with 1% phosphotungstic acid (PTA) adjusted to pH 7.5 with 1N NaOH. OMV production from bacteria grown on solid media was evaluated by picking up and suspending colonies in 50:50 4% paraformaldehyde PBS solution. In addition, a 200-μl fraction of the Brucella broth cell culture was centrifuged at 10000×g for 5min and resuspended in the same volume 4% paraformaldehyde to test the release of OMVs in planktonic conditions. These samples were processed for TEM as explained above. The samples were observed using a Jeol 1200 EX-II transmission electron microscope at 80kV (Jeol Ltd.) at the Service of the Laboratorio Integral de Microscopía, CICVyA, INTA, Argentina. The OMV size and S-layer thickness were expressed as mean±standard deviation. Thickness was calculated with the following formula Thickness=(total diameter−internal diameter)/2. All measurements were conducted by trained staff.Antigenicity of OMVs (Immunodot). An OMV sample from C. fetus 08-421 (80μl) was blotted onto a nitrocellulose membrane using the microfiltration apparatus Bio-Dot (Bio-Rad, Life Science). The same volume of Brucella broth and PBS was used as negative controls and cell proteins of C. fetus were used as positive control. The blot was then blocked with 5% skimmed milk and incubated with a 1:100 dilution of serum anti whole cell proteins of C. fetus. Azul laboratories-Argentina gently donated the C. fetus serum, which was obtained by immunization of rabbits with two C. fetus strains (C. fetus fetus and C. fetus venerealis). After three washes, the blot was incubated with a 1:3000 dilution of alkaline phosphatase-conjugated anti-rabbit IgG (Sigma–Aldrich). After three additional washes, the blot was developed by adding an NBT/BCIP solution (Promega) as substrate.ResultsTo analyze the secretion of OMVs, we first studied the presence of OMVs in bacterium samples grown in solid or broth culture media (non-isolated OMVs). Through TEM, we detected OMVs both surrounding the bacterium cells and detached from cells. The presence of membrane vesicles budding off from the surface of the bacteria was also observed. These non-purified OMVs showed morphological variation (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A). OMVs were also present in cultures grown in solid media (representative image in <a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>E).<elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia>By contrast, the purified OMVs were homogeneous in shape but with variable size, ranging between 24 and 145nm with a mean diameter of 76.58±11.53nm (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>B). Regardless of the subspecies, all the studied strains produced OMVs (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A–D). The analysis of the isolated OMVs revealed the presence of a typical motif of S-layer in all the strains, except for Campylobacter fetus venerealis NCTC 10354, which lacked this crystalline structure (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>D). The absence of the S-layer was also evident in non-purified OMVs from this strain grown on solid media (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>E). S-layer thickness was 15.99±8.91nm (n=20) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>). This finding supports the fact that the S-layer is maintained during the biogenesis of OMVs.<elsevierMultimedia ident="fig0015"></elsevierMultimedia>Finally, a dot-blot immunoblot analysis to assess the antigenicity of released OMVs revealed a strong interaction with C. fetus-hyperimmune rabbit sera in the OMV fraction derived from C. fetus 08-421 (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>).<elsevierMultimedia ident="fig0020"></elsevierMultimedia>DiscussionEarlier electronic microscopy-based studies have provided evidence of the presence of ‘globular bodies’ in Campylobacter fetus, formerly Vibrio fetus<a class="elsevierStyleCrossRef" href="#bib0275">22</a>. Those structures were ignored at that time as OMVs and were characterized as membrane-associated structures that co-purified with flagella<a class="elsevierStyleCrossRef" href="#bib0260">19</a>. Through immunological techniques, Winter and co-workers<a class="elsevierStyleCrossRef" href="#bib0325">32</a> have described the presence of the S-layer protein (former protein A) associated to non-spontaneously produced vesicles obtained by shearing of bacteria. Apart from these studies, no additional evidence of secretion of OMVs nor their isolation have been reported in C. fetus to date.In this work, all the evaluated C. fetus field isolates produced OMVs in vitro and we were able to purify these vesicles successfully through standardized protocols. The shape and sizes obtained were similar to those previously described in other gram-negative bacteria<a class="elsevierStyleCrossRef" href="#bib0255">18</a>. However, it should be considered that OMV sizes could even be more variable (as shown in <a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A); however, the filtration step of the purification process may have led to the elimination of larger OMVs.A characteristic S-layer was evident on the surface of the OMVs in almost all the assayed strains (3/4). High-resolution electron microscopy and scanning probe microscopy have revealed that most S-layers are 5–25nm thick<a class="elsevierStyleCrossRef" href="#bib0285">24</a>, which coincides with our observations by TEM.The S-layer is one of the first bacterial components to interact with the host. Examples of its different roles are cell adhesion, protection from feeding by protozoa or phagocytes, virulence factor, antigenic properties, anchoring sites for hydrolytic exoenzymes, receptors for phages, porin function and others<a class="elsevierStyleCrossRefs" href="#bib0175">2,9</a>. This crystalline structure also provides the bacterium with a protective core against environmental conditions. Hence, some of these functions may also be valid for OMVs, e.g., increasing the lifetime of these transporters to reach distant targets.In addition, the S-layer was absent or undetectable only in OMVs from C. fetus venerealis NCTC 10354. This result was observed both in culture and after purification from cell-free culture media. Interestingly, Blaser and Pei<a class="elsevierStyleCrossRef" href="#bib0185">4</a> described spontaneous S-layer mutants obtained after multiple passages of a wild-type C. fetus strain on artificial media. That could be the case of the C. fetus venerealis NCTC 10354 strain. This strain probably expresses SLPs less efficiently, may not express them at all or may even express SLPs but the process of S-layer assembly could be impaired. Further research must be conducted in relation to the secretion of layered or non-layered OMVs. Thus, considerable attention should be paid when using this strain in future studies, especially when the presence of the S-layer is desirable. The presence of S-layered vesicles is relevant and could have important consequences such as the adhesion to cells or extracellular matrix and modulation of the host response.To test the presence of immunoreactive components, we assayed the recognition of polyclonal sera raised with C. fetus. In our study, C. fetus 08-421 was the most active OMV-producing strain. Despite this fact, we had to employ a dot-blot assay to concentrate on a spot the low quantity of proteins present in the sample. Surface layer proteins have been previously described as important antigens in C. fetus<a class="elsevierStyleCrossRef" href="#bib0180">3</a>. Hence, the immunoreactivity of OMVs that we observed could be explained, in part, by the presence of the S-layer. Additionally, since we cannot discard that polyclonal antibodies could reach the inside of OMVs, other antigenic molecules could also contribute to this observed immunoreactivity. Moreover, surface layer proteins of different pathogens have been described as a pathogen-associated molecular pattern (PAMP). These PAMPs could be recognized by toll-like receptors (TLR) or ICAM-3-grabbing nonintegrin (DC-SIGN) present on macrophages and, in the case of DC-SIGN, on dendritic cells, thus resulting in cell activation and cytokine secretion<a class="elsevierStyleCrossRefs" href="#bib0220">11,13</a>. Therefore, OMVs were described as structures capable of engaging innate and adaptive immunity and with intrinsic adjuvant capacity<a class="elsevierStyleCrossRef" href="#bib0300">27</a>. In this way, C. fetus OMVs could also fulfil important criteria to modulate both adaptive and innate immune responses and constitute a potential candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field.It is worth noting that obtaining OMVs is challenging since their productivity is low. Different culturing times, static growing vs. shacking, volume of culture, initial inoculum as well as different culture media were among the variables assayed to establish the final protocol. The purpose was to obtain native OMVs, thereby the low yields obtained with most of the strains were aligned with the expected results.Stimulated secretion of OMVs could improve the obtained yields. Different stimuli such as nutrient depletion, exposure to antibiotics, hydrogen peroxide, heat shock or even detergent extraction, among others, have been proved to be useful in different bacteria<a class="elsevierStyleCrossRefs" href="#bib0250">17,33</a> and must be evaluated in C. fetus. However, it must be taken into account that the composition of the OMVs could be different<a class="elsevierStyleCrossRef" href="#bib0330">33</a>. For instance, the S-layer could be affected, which could have a detrimental effect on adhesion and immunogenicity.In line with Thompson et al.<a class="elsevierStyleCrossRef" href="#bib0305">28</a>, we think that the engineering of OMVs producing-bacteria to generate OMVs is a promising strategy. Through this, a reduced linkage of the outer membrane to the peptidoglycan can increase OMVs release<a class="elsevierStyleCrossRef" href="#bib0240">15</a> or could transform them in effective carriers of useful molecules<a class="elsevierStyleCrossRef" href="#bib0170">1</a> (antigens, immunological mediators such as cytokines, among others) and even with a controlled host response (e.g., low LPS content)<a class="elsevierStyleCrossRef" href="#bib0330">33</a>. Consequently, its combination with vectored or traditional vaccines could be the best option to boost an overall immunological response.Future proteomic studies could certainly contribute to determining the protein composition and roles of these nanoparticles in C. fetus. This study represents a starting point for future research in C. fetus focused on elucidating the host-pathogen interphase, the modulation of the host response and vaccine development.Conflict of interestThe authors declare that they have no conflicts of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:13 [ 0 => array:3 [ "identificador" => "xres1733197" "titulo" => "Graphical abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:3 [ "identificador" => "xres1733195" "titulo" => "Highlights" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 2 => array:3 [ "identificador" => "xres1733196" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0015" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec1529461" "titulo" => "Keywords" ] 4 => array:3 [ "identificador" => "xres1733198" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0020" ] ] ] 5 => array:2 [ "identificador" => "xpalclavsec1529462" "titulo" => "Palabras clave" ] 6 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 7 => array:2 [ "identificador" => "sec0010" "titulo" => "Material and methods" ] 8 => array:2 [ "identificador" => "sec0015" "titulo" => "Results" ] 9 => array:2 [ "identificador" => "sec0020" "titulo" => "Discussion" ] 10 => array:2 [ "identificador" => "sec0025" "titulo" => "Conflict of interest" ] 11 => array:2 [ "identificador" => "xack611776" "titulo" => "Acknowledgments" ] 12 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2020-11-18" "fechaAceptado" => "2021-06-07" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1529461" "palabras" => array:4 [ 0 => "Outer membrane vesicles" 1 => "Campylobacter" 2 => "Bacterial pathogen" 3 => "Virulence" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec1529462" "palabras" => array:4 [ 0 => "Vesículas de membrana externa" 1 => "Campylobacter" 2 => "Patógeno bacteriano" 3 => "Virulencia" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "The study of outer membrane vesicles (OMVs) became relevant because of their probable important role in the transfer of virulence factors to host cells. Campylobacter fetus is mainly a mammal pathogen whose virulence characterization is still limited. The aim of this study was to evaluate and to characterize the secretion of OMVs in this bacterium. By transmission electron microscopy, we confirmed the production of OMVs in all the strains assayed. Purified OMVs showed a spherical shape and variable size, although comparable to those of other gram-negative bacteria. We also confirmed the presence of the S-layer on the surface of the OMVs of all the strains assayed with the exception of those derived from the NTCC reference strain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C. fetus OMVs could contribute to the modulation of the host response and constitute a candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field. This work represents a platform to drive future studies towards the role of these subcellular structures in C. fetus-host interaction." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "El estudio de las vesículas de membrana externa (VME) tomó un rol protagónico, ya que se las ha relacionado con la transferencia de factores de virulencia a la célula hospedadora. Campylobacter fetus es, principalmente, un patógeno de mamíferos cuya virulencia solo ha sido caracterizada de forma limitada. El objetivo de este trabajo fue evaluar y caracterizar la secreción de VME en esta bacteria. Mediante microscopía electrónica de transmisión confirmamos la producción espontánea de VME en todas las cepas estudiadas. Las VME purificadas mostraron una morfología esférica y un tamaño variable, pero compatible con el reporte de otras bacterias gram negativas. Asimismo, hemos demostrado que estas vesículas conservan la capa S en todas las cepas, menos en la cepa de referencia NCTC y hemos confirmado su inmunorreactividad por dot-blot inmunoblot. Estas VME de C. fetus podrían contribuir a la modulación de la respuesta del hospedador y constituir un buen candidato como adyuvante de las actuales vacunas empleadas en el campo veterinario. Este trabajo representa una plataforma para impulsar estudios futuros en torno al rol de estas estructuras subcelulares en la interfase C. fetus-hospedador." ] ] "highlights" => array:2 [ "titulo" => "Highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•Campylobacter fetus produces spontaneously outer membrane vesicles in vitro.</li><li class="elsevierStyleListItem" id="lsti0010">•S-layer is preserved on the surface of the outer membrane vesicles of C. fetus.</li><li class="elsevierStyleListItem" id="lsti0015">•C. fetus-outer membrane vesicles are antigenic structures that could modulate the immune response.</li><li class="elsevierStyleListItem" id="lsti0020">•C. fetus-outer membrane vesicles can be considered as good candidates for vaccine design.</li></ul>" ] "multimedia" => array:5 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2699 "Ancho" => 2917 "Tamanyo" => 417126 ] ] "descripcion" => array:1 [ "en" => "Release and purification of C. fetus OMVs. (A) Non-purified OMVs (TEM micrograph, negative staining): bacterial fraction from C. fetus 08-421 culture grown in Brucella broth. A flagellate bacterium cell is surrounded by OMVs. The arrows indicate OMVs of different size and shape. (B) and (C) morphometry of purified OMVs. TEM micrographs of negative stained C. fetus 08-421 showing spherical OMVs covering a wide range of sizes (range 26–141nm), mean diameter: 76.58±11.53nm (n=244 from three micrographs)." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1944 "Ancho" => 2917 "Tamanyo" => 359518 ] ] "descripcion" => array:1 [ "en" => "OMVs from different strains of C. fetus. (A) C. fetus fetus 08-421, (B) C. fetus fetus 13-344, (C) C. fetus venerealis biovar intermedius 06-341, (D) C. fetus venerealis NCTC 10354 and (E) C. fetus venerealis NCTC 10354 (non-purified OMVs)." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1370 "Ancho" => 2500 "Tamanyo" => 159078 ] ] "descripcion" => array:1 [ "en" => "S-layer. (A) Morphometry of a representative OMV from C. fetus venerealis biovar intermedius 06-341. The arrowhead shows the S-layer observed as the outermost surface. (B) Schematic representation of an OMV surrounded by the S-layer." ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 421 "Ancho" => 1750 "Tamanyo" => 21492 ] ] "descripcion" => array:1 [ "en" => "Evaluation of the immunoreactivity of OMVs by dot-immunoblot. 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Campylobacter fetus secreta vesículas de membrana externa inmunorreactivas que conservan la capa S

Pablo Faracea, Silvio Craveroa, Catalina Taibob, Julián Diodatib, Claudia Morsellac, Fernando Paolicchic, Julia Sabio y Garcíaa,b, Andrea Gioffréa,

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gioffre.andrea@inta.gob.ar

Corresponding author.

a Instituto de Agrobiotecnología y Biología Molecular, INTA-CONICET, CICVyA-CNIA, Hurlingham, Buenos Aires, Argentina

b Laboratorio Integral de Microscopía, CICVyA, INTA Hurlingham, Buenos Aires, Argentina

c Laboratorio de Bacteriología-Departamento de Sanidad Animal EEA-INTA Balcarce, Buenos Aires, Argentina

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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introduction</span><p id="par0025" class="elsevierStylePara elsevierViewall">The biogenesis and release of outer membrane vesicles &#40;OMVs&#41; is a common feature of several gram-negative bacteria&#46; Through the release of OMVs and the subsequent fusion of membranes&#44; different bacterial components could be delivered and incorporated into the host cells&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">Since their first description in the literature&#44; OMVs have been associated to relevant functions related to host-pathogen &#40;and bacteria&#8211;bacteria&#41; interaction&#44; such as delivery of virulence factors and immune modulators&#44; disruption of host tissue&#44; gene and RNA transfer among others<a class="elsevierStyleCrossRefs" href="#bib0235"><span class="elsevierStyleSup">14&#44;25</span></a>&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Campylobacter</span> are microaerophilic&#44; motile&#44; non-fermentative gram-negative organisms&#46; Most species of <span class="elsevierStyleItalic">Campylobacter</span> can cause disease in both humans and animals&#46; <span class="elsevierStyleItalic">Campylobacter fetus</span> comprises two subspecies associated to mammal hosts &#40;<span class="elsevierStyleItalic">Campylobacter fetus fetus</span> and <span class="elsevierStyleItalic">Campylobacter fetus venerealis</span>&#41;&#46; In animals&#44; they have been associated to bovine infertility and bovine&#47;ovine abortion<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">21</span></a>&#46; <span class="elsevierStyleItalic">C&#46; fetus fetus</span> has been isolated from blood&#44; abscess fluid&#44; bone marrow&#44; cerebrospinal fluid&#44; joint prostheses or implanted catheters and biopsies or stool from humans with different clinical conditions such as fever&#44; colitis&#44; venous thrombosis&#44; erysipelas&#44; meningitis&#44; pleuritic chest pain&#44; lethargy&#44; endocarditis&#44; photophobia and gastrointestinal problems<a class="elsevierStyleCrossRefs" href="#bib0205"><span class="elsevierStyleSup">8&#44;31</span></a>&#46;</p><p id="par0040" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Campylobacter</span> spp&#46; have an outermost surface component&#44; which is called the surface layer &#40;S-layer&#41;&#44; which consists of a crystalline array of proteinaceous subunits &#40;SLP or surface layer proteins&#41;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">26</span></a> encoded by a diverse number of homologous genes in each strain<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">29</span></a>&#46; In <span class="elsevierStyleItalic">C&#46; fetus</span>&#44; SLPs attach to either type A or B lipopolysaccharides<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">28</span></a>&#46; This bidimensional structure&#44; which is external to the cell wall&#44; fulfils a variety of biological functions and roles<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">9</span></a>&#46; The S-layer has been characterized as a virulence factor that mediates the evasion of the immune response in <span class="elsevierStyleItalic">C&#46; fetus</span><a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">3&#44;4</span></a>&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall">Different virulence strategies have been widely described in the emblematic member of the genus&#58; <span class="elsevierStyleItalic">C&#46; jejuni</span>&#46; One of the most innovative strategies is the delivery of the biologically active toxin CDT through OMVs<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">16</span></a>&#46; While <span class="elsevierStyleItalic">C&#46; jejuni</span> virulence has been widely studied&#44; the description of <span class="elsevierStyleItalic">C&#46; fetus</span>-host interphase has been delayed&#46; In addition&#44; OMVs have immunological properties that are suitable for vaccine strategies<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">27</span></a>&#46; Interestingly&#44; a <span class="elsevierStyleItalic">Neisseria meningitidis</span> OMV-based vaccine&#44; the first licensed vaccine based on these nanoparticles&#44; has demonstrated its usefulness in human health<a class="elsevierStyleCrossRefs" href="#bib0225"><span class="elsevierStyleSup">12&#44;23</span></a>&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">In Argentina&#44; reproductive diseases impair livestock production and <span class="elsevierStyleItalic">C&#46; fetus</span> is among the main bacterial pathogens isolated from abortions in one of the most productive regions of the country<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">20</span></a>&#46; <span class="elsevierStyleItalic">C&#46; fetus</span> is the etiological agent of the venereal disease called bovine genital campylobacteriosis&#46; Vaccination is not compulsive&#59; however&#44; it is often applied when positive animals are detected in the herd&#46; The limited available data around bacterin-based commercial vaccines suggest that they must be improved<a class="elsevierStyleCrossRefs" href="#bib0190"><span class="elsevierStyleSup">5&#44;7</span></a>&#46; The use of novel adjuvants could improve the accuracy of current vaccines&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall">The aim of this study was to evaluate the biogenesis of OMVs in <span class="elsevierStyleItalic">C&#46; fetus</span>&#44; to characterize them morphometrically and to assay their immunoreactivity&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Material and methods</span><p id="par0060" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Bacterial strains</span>&#46; <span class="elsevierStyleItalic">Campylobacter fetus</span> strains from the culture collection of the Bacteriology laboratory EEA-INTA Balcarce were used in this study&#46; Three well-characterized <span class="elsevierStyleItalic">C&#46; fetus</span> field strains obtained from tissue samples from bovine aborted fetuses were analyzed in the study&#58; <span class="elsevierStyleItalic">Campylobacter</span><span class="elsevierStyleItalic">fetus venerealis</span> biovar intermedius 06-341 &#40;Buenos Aires&#41;&#44; <span class="elsevierStyleItalic">Campylobacter</span><span class="elsevierStyleItalic">fetus fetus</span> 13-344 &#40;Buenos Aires&#41;&#44; <span class="elsevierStyleItalic">C&#46; fetus fetus</span> 08-421 &#40;Santa Fe&#41;&#46; The type strain <span class="elsevierStyleItalic">C&#46; fetus venerealis</span> NCTC 10354 isolated from bovine vaginal mucus was added in the analyses&#46; We have previously typed the strains following standard biochemical procedures<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">21</span></a>&#44; whereas other studies have characterized them at molecular and genomic levels as well<a class="elsevierStyleCrossRefs" href="#bib0215"><span class="elsevierStyleSup">10&#44;30</span></a>&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Growth conditions and isolation of OMVs</span>&#46; OMVs were obtained by differential centrifugation from bacterial culture supernatants essentially as previously described by Elmi and co-workers for <span class="elsevierStyleItalic">C&#46; jejuni</span> with some modifications<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">6</span></a>&#46; Skirrow agar plates &#40;Oxoid&#41; were used for the recovery of the <span class="elsevierStyleItalic">C&#46;</span><span class="elsevierStyleItalic">fetus</span> strains&#46; Strains were grown at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 72<span class="elsevierStyleHsp" style=""></span>h &#40;5&#37; O<span class="elsevierStyleInf">2</span>&#44; 10&#37; CO<span class="elsevierStyleInf">2</span> and 85&#37; N<span class="elsevierStyleInf">2</span>&#41; and then the colonies were inoculated into 10<span class="elsevierStyleHsp" style=""></span>ml <span class="elsevierStyleItalic">Brucella</span> broth &#40;Oxoid&#41; &#40;starter culture&#41;&#46; The cultures were grown on an orbital shaker &#40;100<span class="elsevierStyleHsp" style=""></span>rpm&#41; under microaerophilic conditions at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 72<span class="elsevierStyleHsp" style=""></span>h&#46; Then&#44; the starter culture was transferred to a flask containing 500<span class="elsevierStyleHsp" style=""></span>ml <span class="elsevierStyleItalic">Brucella</span> broth&#46; Because <span class="elsevierStyleItalic">C&#46; fetus</span> is a slow growing bacterium&#44; long-term cultures were grown to obtain the appropriate optical density &#40;OD 0&#46;3&#8211;0&#46;4 reached after 72<span class="elsevierStyleHsp" style=""></span>h&#8211;96<span class="elsevierStyleHsp" style=""></span>h of culture&#41;&#46; The bacterial culture was centrifuged at 10<span class="elsevierStyleHsp" style=""></span>000<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 30<span class="elsevierStyleHsp" style=""></span>min and the resulting supernatants were filtered through a 0&#46;22<span class="elsevierStyleHsp" style=""></span>&#956;m membrane &#40;Millipore&#41; to remove cells and debris&#46; Then&#44; ultracentrifugation was carried out at 150<span class="elsevierStyleHsp" style=""></span>000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 3<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C using a 45Ti rotor &#40;Beckman instruments&#41; and the pellet containing OMVs was resuspended with a minimal volume of sterile phosphate-buffered saline pH 7&#46;4 &#40;PBS&#41;&#46; Two further washing steps were performed with PBS before carrying out the immunoassays&#46;</p><p id="par0070" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Transmission electron microscopy &#40;TEM&#41; and morphometry</span>&#46; The OMV fraction was fixed with one volume of 4&#37; paraformaldehyde&#44; mounted on grids and negatively stained with 1&#37; phosphotungstic acid &#40;PTA&#41; adjusted to pH 7&#46;5 with 1<span class="elsevierStyleHsp" style=""></span>N NaOH&#46; OMV production from bacteria grown on solid media was evaluated by picking up and suspending colonies in 50&#58;50 4&#37; paraformaldehyde PBS solution&#46; In addition&#44; a 200-&#956;l fraction of the <span class="elsevierStyleItalic">Brucella</span> broth cell culture was centrifuged at 10<span class="elsevierStyleHsp" style=""></span>000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 5<span class="elsevierStyleHsp" style=""></span>min and resuspended in the same volume 4&#37; paraformaldehyde to test the release of OMVs in planktonic conditions&#46; These samples were processed for TEM as explained above&#46; The samples were observed using a Jeol 1200 EX-II transmission electron microscope at 80<span class="elsevierStyleHsp" style=""></span>kV &#40;Jeol Ltd&#46;&#41; at the Service of the Laboratorio Integral de Microscop&#237;a&#44; CICVyA&#44; INTA&#44; Argentina&#46; The OMV size and S-layer thickness were expressed as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; Thickness was calculated with the following formula Thickness<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#40;total diameter<span class="elsevierStyleHsp" style=""></span>&#8722;<span class="elsevierStyleHsp" style=""></span>internal diameter&#41;&#47;2&#46; All measurements were conducted by trained staff&#46;</p><p id="par0075" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Antigenicity of OMVs &#40;Immunodot&#41;</span>&#46; An OMV sample from <span class="elsevierStyleItalic">C&#46; fetus</span> 08-421 &#40;80<span class="elsevierStyleHsp" style=""></span>&#956;l&#41; was blotted onto a nitrocellulose membrane using the microfiltration apparatus Bio-Dot &#40;Bio-Rad&#44; Life Science&#41;&#46; The same volume of <span class="elsevierStyleItalic">Brucella</span> broth and PBS was used as negative controls and cell proteins of <span class="elsevierStyleItalic">C&#46; fetus</span> were used as positive control&#46; The blot was then blocked with 5&#37; skimmed milk and incubated with a 1&#58;100 dilution of serum anti whole cell proteins of <span class="elsevierStyleItalic">C&#46; fetus</span>&#46; Azul laboratories-Argentina gently donated the <span class="elsevierStyleItalic">C&#46; fetus</span> serum&#44; which was obtained by immunization of rabbits with two <span class="elsevierStyleItalic">C&#46; fetus</span> strains &#40;<span class="elsevierStyleItalic">C&#46; fetus fetus</span> and <span class="elsevierStyleItalic">C&#46; fetus venerealis</span>&#41;&#46; After three washes&#44; the blot was incubated with a 1&#58;3000 dilution of alkaline phosphatase-conjugated anti-rabbit IgG &#40;Sigma&#8211;Aldrich&#41;&#46; After three additional washes&#44; the blot was developed by adding an NBT&#47;BCIP solution &#40;Promega&#41; as substrate&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Results</span><p id="par0080" class="elsevierStylePara elsevierViewall">To analyze the secretion of OMVs&#44; we first studied the presence of OMVs in bacterium samples grown in solid or broth culture media &#40;non-isolated OMVs&#41;&#46; Through TEM&#44; we detected OMVs both surrounding the bacterium cells and detached from cells&#46; The presence of membrane vesicles budding off from the surface of the bacteria was also observed&#46; These non-purified OMVs showed morphological variation &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41;&#46; OMVs were also present in cultures grown in solid media &#40;representative image in <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>E&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0085" class="elsevierStylePara elsevierViewall">By contrast&#44; the purified OMVs were homogeneous in shape but with variable size&#44; ranging between 24 and 145<span class="elsevierStyleHsp" style=""></span>nm with a mean diameter of 76&#46;58<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>11&#46;53<span class="elsevierStyleHsp" style=""></span>nm &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>B&#41;&#46; Regardless of the subspecies&#44; all the studied strains produced OMVs &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A&#8211;D&#41;&#46; The analysis of the isolated OMVs revealed the presence of a typical motif of S-layer in all the strains&#44; except for <span class="elsevierStyleItalic">Campylobacter fetus venerealis</span> NCTC 10354&#44; which lacked this crystalline structure &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>D&#41;&#46; The absence of the S-layer was also evident in non-purified OMVs from this strain grown on solid media &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>E&#41;&#46; S-layer thickness was 15&#46;99<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>8&#46;91<span class="elsevierStyleHsp" style=""></span>nm &#40;n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>20&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46; This finding supports the fact that the S-layer is maintained during the biogenesis of OMVs&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0090" class="elsevierStylePara elsevierViewall">Finally&#44; a dot-blot immunoblot analysis to assess the antigenicity of released OMVs revealed a strong interaction with <span class="elsevierStyleItalic">C&#46; fetus</span>-hyperimmune rabbit sera in the OMV fraction derived from <span class="elsevierStyleItalic">C&#46; fetus</span> 08-421 &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Discussion</span><p id="par0095" class="elsevierStylePara elsevierViewall">Earlier electronic microscopy-based studies have provided evidence of the presence of &#8216;globular bodies&#8217; in <span class="elsevierStyleItalic">Campylobacter fetus</span>&#44; formerly <span class="elsevierStyleItalic">Vibrio fetus</span><a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">22</span></a>&#46; Those structures were ignored at that time as OMVs and were characterized as membrane-associated structures that co-purified with flagella<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">19</span></a>&#46; Through immunological techniques&#44; Winter and co-workers<a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">32</span></a> have described the presence of the S-layer protein &#40;former protein A&#41; associated to non-spontaneously produced vesicles obtained by shearing of bacteria&#46; Apart from these studies&#44; no additional evidence of secretion of OMVs nor their isolation have been reported in <span class="elsevierStyleItalic">C&#46; fetus</span> to date&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">In this work&#44; all the evaluated <span class="elsevierStyleItalic">C&#46; fetus</span> field isolates produced OMVs <span class="elsevierStyleItalic">in vitro</span> and we were able to purify these vesicles successfully through standardized protocols&#46; The shape and sizes obtained were similar to those previously described in other gram-negative bacteria<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">18</span></a>&#46; However&#44; it should be considered that OMV sizes could even be more variable &#40;as shown in <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41;&#59; however&#44; the filtration step of the purification process may have led to the elimination of larger OMVs&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">A characteristic S-layer was evident on the surface of the OMVs in almost all the assayed strains &#40;3&#47;4&#41;&#46; High-resolution electron microscopy and scanning probe microscopy have revealed that most S-layers are 5&#8211;25<span class="elsevierStyleHsp" style=""></span>nm thick<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">24</span></a>&#44; which coincides with our observations by TEM&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">The S-layer is one of the first bacterial components to interact with the host&#46; Examples of its different roles are cell adhesion&#44; protection from feeding by protozoa or phagocytes&#44; virulence factor&#44; antigenic properties&#44; anchoring sites for hydrolytic exoenzymes&#44; receptors for phages&#44; porin function and others<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">2&#44;9</span></a>&#46; This crystalline structure also provides the bacterium with a protective core against environmental conditions&#46; Hence&#44; some of these functions may also be valid for OMVs&#44; <span class="elsevierStyleItalic">e&#46;g</span>&#46;&#44; increasing the lifetime of these transporters to reach distant targets&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">In addition&#44; the S-layer was absent or undetectable only in OMVs from <span class="elsevierStyleItalic">C&#46; fetus venerealis</span> NCTC 10354&#46; This result was observed both in culture and after purification from cell-free culture media&#46; Interestingly&#44; Blaser and Pei<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">4</span></a> described spontaneous S-layer mutants obtained after multiple passages of a wild-type <span class="elsevierStyleItalic">C&#46; fetus</span> strain on artificial media&#46; That could be the case of the <span class="elsevierStyleItalic">C&#46; fetus venerealis</span> NCTC 10354 strain&#46; This strain probably expresses SLPs less efficiently&#44; may not express them at all or may even express SLPs but the process of S-layer assembly could be impaired&#46; Further research must be conducted in relation to the secretion of layered or non-layered OMVs&#46; Thus&#44; considerable attention should be paid when using this strain in future studies&#44; especially when the presence of the S-layer is desirable&#46; The presence of S-layered vesicles is relevant and could have important consequences such as the adhesion to cells or extracellular matrix and modulation of the host response&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">To test the presence of immunoreactive components&#44; we assayed the recognition of polyclonal sera raised with <span class="elsevierStyleItalic">C&#46; fetus</span>&#46; In our study&#44; <span class="elsevierStyleItalic">C&#46; fetus</span> 08-421 was the most active OMV-producing strain&#46; Despite this fact&#44; we had to employ a dot-blot assay to concentrate on a spot the low quantity of proteins present in the sample&#46; Surface layer proteins have been previously described as important antigens in <span class="elsevierStyleItalic">C&#46; fetus</span><a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">3</span></a>&#46; Hence&#44; the immunoreactivity of OMVs that we observed could be explained&#44; in part&#44; by the presence of the S-layer&#46; Additionally&#44; since we cannot discard that polyclonal antibodies could reach the inside of OMVs&#44; other antigenic molecules could also contribute to this observed immunoreactivity&#46; Moreover&#44; surface layer proteins of different pathogens have been described as a pathogen-associated molecular pattern &#40;PAMP&#41;&#46; These PAMPs could be recognized by toll-like receptors &#40;TLR&#41; or ICAM-3-grabbing nonintegrin &#40;DC-SIGN&#41; present on macrophages and&#44; in the case of DC-SIGN&#44; on dendritic cells&#44; thus resulting in cell activation and cytokine secretion<a class="elsevierStyleCrossRefs" href="#bib0220"><span class="elsevierStyleSup">11&#44;13</span></a>&#46; Therefore&#44; OMVs were described as structures capable of engaging innate and adaptive immunity and with intrinsic adjuvant capacity<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">27</span></a>&#46; In this way&#44; <span class="elsevierStyleItalic">C&#46; fetus</span> OMVs could also fulfil important criteria to modulate both adaptive and innate immune responses and constitute a potential candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">It is worth noting that obtaining OMVs is challenging since their productivity is low&#46; Different culturing times&#44; static growing vs&#46; shacking&#44; volume of culture&#44; initial inoculum as well as different culture media were among the variables assayed to establish the final protocol&#46; The purpose was to obtain native OMVs&#44; thereby the low yields obtained with most of the strains were aligned with the expected results&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">Stimulated secretion of OMVs could improve the obtained yields&#46; Different stimuli such as nutrient depletion&#44; exposure to antibiotics&#44; hydrogen peroxide&#44; heat shock or even detergent extraction&#44; among others&#44; have been proved to be useful in different bacteria<a class="elsevierStyleCrossRefs" href="#bib0250"><span class="elsevierStyleSup">17&#44;33</span></a> and must be evaluated in <span class="elsevierStyleItalic">C&#46; fetus</span>&#46; However&#44; it must be taken into account that the composition of the OMVs could be different<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">33</span></a>&#46; For instance&#44; the S-layer could be affected&#44; which could have a detrimental effect on adhesion and immunogenicity&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">In line with Thompson et al&#46;<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">28</span></a>&#44; we think that the engineering of OMVs producing-bacteria to generate OMVs is a promising strategy&#46; Through this&#44; a reduced linkage of the outer membrane to the peptidoglycan can increase OMVs release<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">15</span></a> or could transform them in effective carriers of useful molecules<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">1</span></a> &#40;antigens&#44; immunological mediators such as cytokines&#44; among others&#41; and even with a controlled host response &#40;<span class="elsevierStyleItalic">e&#46;g</span>&#46;&#44; low LPS content&#41;<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">33</span></a>&#46; Consequently&#44; its combination with vectored or traditional vaccines could be the best option to boost an overall immunological response&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">Future proteomic studies could certainly contribute to determining the protein composition and roles of these nanoparticles in <span class="elsevierStyleItalic">C&#46; fetus</span>&#46; This study represents a starting point for future research in <span class="elsevierStyleItalic">C&#46; fetus</span> focused on elucidating the host-pathogen interphase&#44; the modulation of the host response and vaccine development&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Conflict of interest</span><p id="par0145" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
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            0 => "Outer membrane vesicles"
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        "resumen" => "<span id="abst0015" class="elsevierStyleSection elsevierViewall"><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The study of outer membrane vesicles &#40;OMVs&#41; became relevant because of their probable important role in the transfer of virulence factors to host cells&#46; <span class="elsevierStyleItalic">Campylobacter fetus</span> is mainly a mammal pathogen whose virulence characterization is still limited&#46; The aim of this study was to evaluate and to characterize the secretion of OMVs in this bacterium&#46; By transmission electron microscopy&#44; we confirmed the production of OMVs in all the strains assayed&#46; Purified OMVs showed a spherical shape and variable size&#44; although comparable to those of other gram-negative bacteria&#46; We also confirmed the presence of the S-layer on the surface of the OMVs of all the strains assayed with the exception of those derived from the NTCC reference strain&#46; In addition&#44; we demonstrated their immunoreactivity by the dot-blot assay&#46; Hence&#44; <span class="elsevierStyleItalic">C&#46; fetus</span> OMVs could contribute to the modulation of the host response and constitute a candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field&#46; This work represents a platform to drive future studies towards the role of these subcellular structures in <span class="elsevierStyleItalic">C&#46; fetus</span>-host interaction&#46;</p></span>"
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        "resumen" => "<span id="abst0020" class="elsevierStyleSection elsevierViewall"><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">El estudio de las ves&#237;culas de membrana externa &#40;VME&#41; tom&#243; un rol protag&#243;nico&#44; ya que se las ha relacionado con la transferencia de factores de virulencia a la c&#233;lula hospedadora&#46; <span class="elsevierStyleItalic">Campylobacter fetus</span> es&#44; principalmente&#44; un pat&#243;geno de mam&#237;feros cuya virulencia solo ha sido caracterizada de forma limitada&#46; El objetivo de este trabajo fue evaluar y caracterizar la secreci&#243;n de VME en esta bacteria&#46; Mediante microscop&#237;a electr&#243;nica de transmisi&#243;n confirmamos la producci&#243;n espont&#225;nea de VME en todas las cepas estudiadas&#46; Las VME purificadas mostraron una morfolog&#237;a esf&#233;rica y un tama&#241;o variable&#44; pero compatible con el reporte de otras bacterias gram negativas&#46; Asimismo&#44; hemos demostrado que estas ves&#237;culas conservan la capa S en todas las cepas&#44; menos en la cepa de referencia NCTC y hemos confirmado su inmunorreactividad por <span class="elsevierStyleItalic">dot-blot</span> inmunoblot&#46; Estas VME de <span class="elsevierStyleItalic">C&#46; fetus</span> podr&#237;an contribuir a la modulaci&#243;n de la respuesta del hospedador y constituir un buen candidato como adyuvante de las actuales vacunas empleadas en el campo veterinario&#46; Este trabajo representa una plataforma para impulsar estudios futuros en torno al rol de estas estructuras subcelulares en la interfase <span class="elsevierStyleItalic">C&#46; fetus</span>-hospedador&#46;</p></span>"
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      "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall"><ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005"><span class="elsevierStyleLabel">&#8226;</span><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Campylobacter fetus</span> produces spontaneously outer membrane vesicles <span class="elsevierStyleItalic">in vitro</span>&#46;</p></li><li class="elsevierStyleListItem" id="lsti0010"><span class="elsevierStyleLabel">&#8226;</span><p id="par0010" class="elsevierStylePara elsevierViewall">S-layer is preserved on the surface of the outer membrane vesicles of <span class="elsevierStyleItalic">C&#46; fetus</span>&#46;</p></li><li class="elsevierStyleListItem" id="lsti0015"><span class="elsevierStyleLabel">&#8226;</span><p id="par0015" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">C&#46; fetus</span>-outer membrane vesicles are antigenic structures that could modulate the immune response&#46;</p></li><li class="elsevierStyleListItem" id="lsti0020"><span class="elsevierStyleLabel">&#8226;</span><p id="par0020" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">C&#46; fetus</span>-outer membrane vesicles can be considered as good candidates for vaccine design&#46;</p></li></ul></p></span>"
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          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Release and purification of <span class="elsevierStyleItalic">C&#46; fetus</span> OMVs&#46; &#40;A&#41; Non-purified OMVs &#40;TEM micrograph&#44; negative staining&#41;&#58; bacterial fraction from <span class="elsevierStyleItalic">C&#46; fetus</span> 08-421 culture grown in <span class="elsevierStyleItalic">Brucella</span> broth&#46; A flagellate bacterium cell is surrounded by OMVs&#46; The arrows indicate OMVs of different size and shape&#46; &#40;B&#41; and &#40;C&#41; morphometry of purified OMVs&#46; TEM micrographs of negative stained <span class="elsevierStyleItalic">C&#46; fetus</span> 08-421 showing spherical OMVs covering a wide range of sizes &#40;range 26&#8211;141<span class="elsevierStyleHsp" style=""></span>nm&#41;&#44; mean diameter&#58; 76&#46;58<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>11&#46;53<span class="elsevierStyleHsp" style=""></span>nm &#40;n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>244 from three micrographs&#41;&#46;</p>"
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                      "titulo" => "A meningococcal outer membrane vesicle vaccine with overexpressed mutant FHbp elicits higher protective antibody responses in infant rhesus macaques than a licensed serogroup B vaccine"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
                            0 => "P&#46;T&#46; Beernink"
                            1 => "V&#46; Vianzon"
                            2 => "L&#46;A&#46; Lewis"
                            3 => "G&#46;R&#46; Moe"
                            4 => "D&#46;M&#46; Granoff"
                          ]
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Article information

ISSN: 03257541
Original language: English

DOI:10.1016/j.ram.2021.06.001

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