RESUMEN

UNLABELLED: Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of >500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the >500-nm condition, since it presents an ∼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described. IMPORTANCE: Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of >500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of ∼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses.

Asunto(s)

Acanthamoeba/virología , Vesículas Citoplasmáticas/virología , Virus Gigantes/fisiología , Internalización del Virus , Animales , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/genética , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplásmico/ultraestructura , Retículo Endoplásmico/virología , Genoma Viral , Virus Gigantes/ultraestructura , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Fagocitosis , Filogenia , Virión/genética , Virión/fisiología , Virión/ultraestructura , Replicación Viral

RESUMEN

BACKGROUND: Escherichia coli is believed to participate in the etiology of Crohn's disease (CD) and possibly of ulcerative colitis (UC), due at least in part to the observed rise in the number of these bacteria in the gut microbiota of CD and UC patients. Nevertheless, it is not fully understood whether this quantitative variation occurs equally throughout the mucosal and luminal spaces of the gut. To assess this question, stools and mucosa biopsies from distinct intestinal sites were cultured aiming at determining their E. coli concentration. The cultures were additionally screened for the presence of some virulence genes of pathogenic E. coli. RESULTS: Analyses of clinical materials from 14 controls (38 biopsies and 14 stools samples), 11 CD (25 biopsies and 11 stools samples) and 7 UC patients (18 biopsies and 7 stools samples) indicated no significant variation in the number of E. coli present in stools, but a rise of at least one log10 CFU/mg in biopsies from the ileum of CD patients and the sigmoid and rectum of CD and UC patients. The cultures were screened for the presence of E. coli attaching and effacing (eae), invasion plasmid antigen H (ipaH), aggregative adherence transcriptional activator (aggR), Shiga cytotoxins (stx), and heat labile enterotoxin (elt) and the following serine proteases autotransporters of Enterobacteriaceae (SPATE) genes: plasmid encoded toxin (pet), secreted autotransporter toxin (sat), Shigella extracellular protein (sepA), protein involved in intestinal colonization (pic) and Shigella IgA-like protease homolog (sigA). Six of the 10 genes screened were detected in the total of samples investigated: aggR, eae, pet, sat, sepA and sigA. No difference in the prevalence of any of these markers was observed in cultures from different clinical materials or groups of patients. METHODS: Bacterial quantitation was carried out following cultures of diluted samples suspensions in MacConkey agar, Wilkins Chalgren agar for anaerobes, E. coli/coliform chromocult agar, and blood agar. Screening for E. coli virulence genes was performed by multiplex PCR of DNA purified from total MacConkey undiluted broth cultures. CONCLUSION: In CD and UC patients only the mucosa associated population of E. coli is augmented and the proliferation is prominent in the ileum of CD and rectum and sigmoid of both UC and CD patients which are sites where the lesions usually are observed. The augmented E. coli population in these sites presented a low number of the virulence markers, possibly meaning that they are not relevant for the disease process.