Characterization of the Na⁺/H⁺ antiporter from Yersinia pestis

Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na/H antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein''s substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein''s structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na/H antiporter''s structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na/H antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.     

Typing of Yersinia pestis isolates from the state of Ceará, Brazil

AIMS: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment. METHODS AND RESULTS: The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971-1997). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.     

Hijacking of the pleiotropic cytokine interferon-γ by the type III secretion system of Yersinia pestis

Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.     

Emergence and Spread of Basal Lineages of Yersinia pestis during the Neolithic Decline

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Loss of heterozygosity in somatic cells of the mouse. An important step in cancer initiation?

Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.     

Isolation and characterization of autoagglutination factor of Yersinia pestis Hms− cells

An approach for isolation of an autoagglutination factor (AF) from Hms(-) cells of the plague agent has been developed. Purified AF has been obtained and characterized in physicochemical properties. The AF is found to be a complex of a 17.5-kD protein with a low molecular weight peptide component, which binds iron ions and shows siderophore activity. This low molecular weight component is responsible for hydrophobic properties and immunochemical activity of the AF, as well as for its ability to interact with the plague diagnosticum L-413c bacteriophage.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Is Toxoplasma egress the first step in invasion?

The protozoan parasite Toxoplasma gondii maintains an intracellular lifestyle that requires careful timing and coordination when exiting one cell (egress) and entering another (invasion). Here it is argued that T. gondii uses similar molecular mechanisms for egress and invasion, based on common morphology, dependence on motility, and regulation by a calcium-dependent signal transduction pathway. In our view, this strategy is highly advantageous because it allows the parasite to egress rapidly from one cell and immediately invade an adjacent cell, thereby minimizing exposure to the extracellular environment where it could be destroyed by host immune mediators.     

Aerosols: An underestimated vehicle for transmission of prion diseases?

We and others have recently reported that prions can be transmitted to mice via aerosols. These reports spurred a lively public discussion on the possible public-health threats represented by prion-containing aerosols. Here we offer our view on the context in which these findings should be placed. On the one hand, the fact that nebulized prions can transmit disease cannot be taken to signify that prions are airborne under natural circumstances. On the other hand, it appears important to underscore the fact that aerosols can originate very easily in a broad variety of experimental and natural environmental conditions. Aerosols are a virtually unavoidable consequence of the handling of fluids; complete prevention of the generation of aerosols is very difficult. While prions have never been found to be transmissible via aerosols under natural conditions, it appears prudent to strive to minimize exposure to potentially prion-infected aerosols whenever the latter may arise—for example in scientific and diagnostic laboratories handling brain matter, cerebrospinal fluids, and other potentially contaminated materials, as well as abattoirs. Equally important is that prion biosafety training be focused on the control of, and protection from, prion-infected aerosols.Key words: prion, prion transmission, scrapie, chronic wasting diseases, CWD, Creutzfeldt-Jacob-disease, CJD, TSE, aerosol, pathogens, allergensPrions, the causative agents of transmissible spongiform encephalopathies, can be undoubtedly propagated from one individual organism to another. The specific routes of prion transmission have been subjected to intensive studies over the past two decades. Incidental and iatrogenic transmission has occurred through the intracerebral route in the case of Dura mater implants¹ and the parenteral route in the case of contaminated pituitary hormones.² In addition, the Bovine Spongiform Encephalopathy (BSE) disaster has provided grim evidence that prion can be transmitted enterally as well. Experimental transmission of prions has been routinely achieved via intraperitoneal and intravenous injection^3,4 but also through more exotic routes such as intralingual,⁵ intranerval⁶ and conjunctival inoculation⁷ and via the nasal cavity.⁸In all prion disease paradigms studied so far the propagation, accumulation and dissemination of the prion protein has been mostly shown to depend on a functional immune system.^9–12 This dependence of prion pathogenesis on the lymphoid compartment, however, is only true for peripheral routes of infection—whereas direct inoculation into the brain does not require any components of the adaptive or innate immune system.B cells in secondary lymphoid organs have been shown to be of importance for the neuroinvasion of the prion protein; in contrast, B lymphocytes in the blood do not appear to play a crucial role.^13–15A special role in prion pathogenesis can be assigned to follicular dendritic cells (FDC). The generation, maturation and function of FDC are dependent on cytokines and chemokines predominantly synthesized and secreted by B lymphocytes. Consistently with this role of B cells in prion pathogenesis, B cell deficient mice show a significantly impaired prion replication due to severely impaired maturation of FDCs.¹⁶ Other soluble and membrane-bound immune mediators such as lymphotoxin heterotrimers and TNFalpha^17,18 as well as components of the complement system^19,20 play an important role in prion pathogenesis.While prions mostly reside in tissues, prion infectivity has also been detected in a variety of body fluids including cerebrospinal fluid,²¹ blood,²² saliva,²³ milk²⁴ and urine.²⁵ Although shedding of prions may occur constitutively from these secretions and excretions, many of the latter phenomena are enhanced by chronic inflammatory processes such as granulomas²⁶ and follicular infiltrates,²⁷ which trigger the maturation of lymphotoxin-dependent, prion-replicating cells.²⁶ The presence of prions in fluids begs the question whether nebulization, and subsequent inhalation, of such fluids may trigger prion infections.Aerosols are finely dispersed particles originating from solid material or liquid using air or other gases as carriers. Natural examples of aerosols include dust (e.g., volcano ashes), smoke, haze and sprays (e.g., sneezing or sea water sprays from breaking waves). Aerosols might be formally categorized as primary or secondary, with primary aerosols being generated in mechanical or thermal processes e.g., by whirling up, impact on surfaces, or burning, whereas secondary aerosols are generated during chemical reactions or by using condensation nuclei.Primary aerosols play an important role in microbiology since they can act as efficacious vehicles for pollen, spores, algae, fungi, bacteria and viruses. Of medical importance are also dandruff, fragments of fur, hairs or skin and mites, which can all function as allergens and trigger e.g., allergic asthma.Moreover, aerosols are excellent vehicles for the transportation of drugs into the respiratory tract. The size of the individual droplets is crucial in specifying the target organs of aerosol. Particle sized 3–10 µm are generally deposited in the nasal cavity and in the throat, whereas smaller particles (e.g., 1 µm) tend to deposit within the lower airways. In rodents pulmonary deposition can reach 10%.^28,29 In humans, particles of 5 µm may reach the lung if inhaled orally, but deposition in the alveolar compartment after inhaling via the nose is highly unlikely.^28,29 For the reasons discussed above, we have become interested in exploring the transmission potential of aerosol-borne prions. Indeed, we found that mouse scrapie can be efficiently transmitted via aerosols.³⁰ In addition to results obtained by exposure to aerosols, we found that mice developed prion infections when inoculated intranasally.Interestingly, this route of transmission was entirely independent on immune cells as shown by challenging various transgenic mouse strains lacking defined functions of the immune system.Well-known examples of transmission of pathogens via aerosols are infections by respiratory viruses (e.g., influenza viruses, adenoviruses, rhinoviruses, coronaviruses) and bacterial diseases (e.g., legionellosis, pneumonic plague by Yersinia pestis, Q-fever by Coxiella burnettii, anthrax) and fungal diseases (particularly aspergillosis and candidosis). In stark contrast, aerosols have historically never been regarded as potential vectors for prion diseases—although very little data existed in favor or against this possibility. This attitude goes along with the implicit “conventional wisdom” that prions are not airborne diseases. However, the concept of “airborne disease” in all the bacterial, fungal and viral examples quoted above, encompasses three distinct phases: (1) release of the infectious agent into aerosols by an infected donor, (2) uptake by a healthy recipient and (3) establishment of disease. It is self-evident that little or no natural transmission between individuals will be observed if any one of these three steps is inefficient. The epidemiological evidence from human prion diseases seems to indicate, albeit indirectly, that step #1 does not occur in CJD patients—inter alia because there is a dearth of evidence of proximity clustering of sCJD.³¹ In the case of CWD the situation may be different since saliva and droppings, which might plausibly give rise to powerful aerosols under a variety of conditions, were found to harbor infectivity. Finally, milk from sheep affected by mastitis can carry scrapie infectivity and—again—could conceivably give rise to aerosols. Since both CWD and sheep scrapie can efficiently spread horizontally within animal collectives, it is extremely appealing to speculate whether aerosols may play a role in said transmission.In natural scrapie in sheep horizontal transmission of prion diseases has been long thought to arise from placental contamination. However, in mice suffering from nephritis prion infectivity is shed with the urine.²⁵ Furthermore, sheep having a mastitis can transmit infectious prions with milk.³²In Chronic Wasting disease (CWD) of deer several careful studies have been performed that, together with our present finding, depose in favor of airborne transmission in this naturally occurring disease. Indeed, CWD prions can be transmitted experimentally via aerosol and the nasal route to transgenic cervidized mice.³³ Although no anecdotal or epidemiological evidence has come forward that airborne transmission may be important for the spread of CWD, several lines of thought suggest that this possibility is not implausible. In deer, prions have been detected in urine, saliva, feces and blood of diseased animals. Moreover, it was claimed that pathological prion protein could be recovered from the environmental water in an endemic area.³⁴ Since all fluids can act as sources for the generation of aerosols, any of the body fluids mentioned above may represent the point of origin for airborne transmission of CWD prions.In this context, also the presence of infectious prions in blood of patients should be mentioned which was demonstrated by the transmission of vCJD by blood transfusions.^35,36 The growing body of evidence that prion transmission can be airborne—at least under certain conditions—dictates that the release of potentially contaminated aerosols should be avoided under all circumstances. In this context it is mandatory that reliable precautions be defined and followed in scientific and diagnostic laboratories. In particular, it is self-evident that safety cabinets should be used while processing brain and nerve tissue (or any other potentially contaminated tissue) of man and animals suspected with prion disease. Our experience shows that this necessity is generally very well-understood by prion scientists.A further stone of contention relates to the biosafety level of the laboratory environment. Because prions were hitherto considered not be airborne, so far no specific regulations have been implemented. As a consequence, prion laboratories have been mostly required to adhere to the category “BSL3**.” While it is understood that the airborne transmission of prions has thus far only been observed under extreme conditions, we feel that it is in order to critically reassess biosafety regulations in the light of the recent discoveries. In particular, one might consider implementing more stringent measures towards protecting workers within diagnostic and scientific laboratories from aerosols.The situation in slaughterhouses and plants handling potentially contaminated offal may be even more problematic. Although regulations in slaughterhouses dictate the use of protecting glasses and masks or, alternatively, visors the use of personal protecting equipment should be rigorously controlled. In addition, high-pressure cleaning devices produce massive aerosols and should be strictly avoided in areas of slaughterhouses where prion-containing material may be processed. Regulations concerning cleaning of heads from slaughtered animals do pay attention to aerosol avoidance, e.g., by allowing only water hoses without pressure.A case in point is the severe neurological syndrome arising in swine abattoir workers.³⁷ Here, an immune-mediated polyradiculoneuropathy was reported to be related to a process using high-pressure fluids to remove the brains of swine.³⁷ During this process, high amounts of swine brain tissue became aerosolized and were inhaled and/or gained access to the respiratory tract mucosa of abattoir workers, resulting in immunization with myelin constituents akin to experimental autoimmune encephalitis (EAE). Although significant physiological differences exist concerning breathing, where humans are regarded as mouth breathers and mice as nose breathers, many people indeed show nose breathing under no or only moderate body burden. Therefore, results obtained in mouse experiments might also be extrapolated to a considerable extent to the situation in man.In this context it is of importance to stress again that aerosols might be generated under various conditions and represent a normal entity of the environment in a variety of daily life situations.In our studies of airborne transmission of prion protein in mice³⁰ we took advantage of the fact that mice breathe exclusively through their nostrils^38,39 and therefore could be exposed in groups to aerosolized brain suspensions. Using this system, it was possible to vary both time of exposure as well as concentration of the prion load in the aerosol. We were surprised to discover that exposure times as short as 1 min were sufficient to achieve high attack rates. By extending the time of exposure it became obvious that incubation times were shortened. A possible alternative route of infection via the cornea or the conjunctiva was extremely unlikely, since newborn mice, whose eyelids were still closed, could also be infected. These findings show that the aerogenic transmission of prions is very efficient.But how do prions spread from the airways to the brain? Peripheral replication of prions in the lymphoid system—a characteristic of most other peripheral routes of transmission—appeared to be dispensable. Instead, the results argue for a direct pathway of brain invasion. One anatomical peculiarity of the nasal cavity is the “area cribriformis” of the olfactory epithelium. Here the olfactory bulb sprouts axons of olfactory receptor neurons passing through the cribriform plate of the ethmoidal bone to reach the olfactory mucosa where olfactory cilia extend representing non-myelinated nerve endings. Thus, open nerve endings are located in the nasal cavity through which aerosolized infectious prions might get access to the brain. In this context it is noteworthy that pathological prion protein was found in the olfactory cilia and basal cells of the olfactory mucosa of sCJD patients, as well as in the olfactory bulb and olfactory tract.^40,41 However, it was hitherto never clearly documented that olfactory receptor neurons represent an entry site for infectious prions; this might also be due to the sensitivity threshold of detection assays.In conclusion, aerosols can infect mice with a surprisingly high efficiency. Just how important a role is played by this newly recognized pathway of spread in natural transmission is, as of now, unclear and in need of further studies. Although it was not identified as a route of infection in epidemiological studies thus far, the worryingly high attack rate suggests that we would be well-advised to carefully avoid the inhalation of aerosols from prion-containing materials.