Yersinia pestis Resists Predation by Acanthamoeba castellanii and Exhibits Prolonged Intracellular Survival.

Plague is a flea-borne rodent-associated zoonotic disease caused by Yersinia pestis The disease is characterized by epizootics with high rodent mortalities, punctuated by interepizootic periods when the bacterium persists in an unknown reservoir. This study investigates the interaction between Y. pestis and the ubiquitous soil free-living amoeba (FLA) Acanthamoeba castellanii to assess if the bacterium can survive within soil amoebae and whether intracellular mechanisms are conserved between infection of mammalian macrophages and soil amoebae. The results demonstrate that during coculture with amoebae, representative Y. pestis strains of epidemic biovars Medievalis, Orientalis, and Antiqua are phagocytized and able to survive within amoebae for at least 5 days. Key Y. pestis determinants of the intracellular interaction of Y. pestis and phagocytic macrophages, PhoP and the type three secretion system (T3SS), were then tested for their roles in the Y. pestis-amoeba interaction. Consistent with a requirement for the PhoP transcriptional activator in the intracellular survival of Y. pestis in macrophages, a PhoP mutant is unable to survive when cocultured with amoebae. Additionally, induction of the T3SS blocks phagocytic uptake of Y. pestis by amoebae, similar to that which occurs during macrophage infection. Electron microscopy revealed that in A. castellanii, Y. pestis resides intact within spacious vacuoles which were characterized using lysosomal trackers as being separated from the lysosomal compartment. This evidence for prolonged survival and subversion of intracellular digestion of Y. pestis within FLA suggests that protozoa may serve as a protective soil reservoir for Y. pestisIMPORTANCEYersinia pestis is a reemerging flea-borne zoonotic disease. Sylvatic plague cycles are characterized by an epizootic period during which the disease spreads rapidly, causing high rodent mortality, and an interepizootic period when the bacterium quiescently persists in an unknown reservoir. An understanding of the ecology of Y. pestis in the context of its persistence in the environment and its reactivation to initiate a new epizootic cycle is key to implementing novel surveillance strategies to more effectively predict and prevent new disease outbreaks. Here, we demonstrate prolonged survival and subversion of intracellular digestion of Y. pestis within a soil free-living amoeba. This suggests the potential role for protozoa as a protective soil reservoir for Y. pestis, which may help explain the recrudescence of plague epizootics.

Yersinia pseudotuberculosis IP32953 survives and replicates in trophozoites and persists in cysts of Acanthamoeba castellanii.

Yersinia pseudotuberculosis is a foodborne enteric pathogen that causes a mild self-limiting diarrhea in humans. Yersinia pseudotuberculosis is able to persist in soil and water and in association with fresh produce, but the mechanism by which it persists is unknown. It has been shown that Y. pseudotuberculosis co-occurs with protozoans in these environments; therefore, this study investigates if bacterivorous free-living amoeba (FLA) are able to support persistence of Y. pseudotuberculosis. Coculture studies of Y. pseudotuberculosis and the prototype FLA, Acanthamoeba castellanii revealed that bacteria had an enhanced capacity to survive in association with amoeba and in the absence of any cytotoxic effects. Yersinia pseudotuberculosis is able to survive and replicate in trophozoites specifically localized within vacuoles, and persists within cysts over a period of at least a week. These data present the first evidence that Y. pseudotuberculosis is able to resist the bacterivorous nature of FLA and instead exhibits an enhanced ability to replicate and persist in coculture with amoeba. This study sheds light on the potential role of FLA in the ecology of Y. pseudotuberculosis which may have implications for food safety.

Unraveling a three-step spatiotemporal mechanism of triggering of receptor-induced Nipah virus fusion and cell entry.

Membrane fusion is essential for entry of the biomedically-important paramyxoviruses into their host cells (viral-cell fusion), and for syncytia formation (cell-cell fusion), often induced by paramyxoviral infections [e.g. those of the deadly Nipah virus (NiV)]. For most paramyxoviruses, membrane fusion requires two viral glycoproteins. Upon receptor binding, the attachment glycoprotein (HN/H/G) triggers the fusion glycoprotein (F) to undergo conformational changes that merge viral and/or cell membranes. However, a significant knowledge gap remains on how HN/H/G couples cell receptor binding to F-triggering. Via interdisciplinary approaches we report the first comprehensive mechanism of NiV membrane fusion triggering, involving three spatiotemporally sequential cell receptor-induced conformational steps in NiV-G: two in the head and one in the stalk. Interestingly, a headless NiV-G mutant was able to trigger NiV-F, and the two head conformational steps were required for the exposure of the stalk domain. Moreover, the headless NiV-G prematurely triggered NiV-F on virions, indicating that the NiV-G head prevents premature triggering of NiV-F on virions by concealing a F-triggering stalk domain until the correct time and place: receptor-binding. Based on these and recent paramyxovirus findings, we present a comprehensive and fundamentally conserved mechanistic model of paramyxovirus membrane fusion triggering and cell entry.

Detection of receptor-induced glycoprotein conformational changes on enveloped virions by using confocal micro-Raman spectroscopy.

Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry.