Primordial Proteins and HIV Andrei P. Sommer* and Attila E. Pavla´ th† Central Institute of Biomedical Engineering, University of Ulm, 89081 Ulm, Germany Received November 11, 2004
Primordial proteins regulate the response of nanobacteria to variations in their environment and reinforce existing pathogenic potentials. By analyzing specific response patterns, we predicted the prevalence of nanobacteria in HIVsand in the atmosphere. A current clinical study indicates the identification of a possibly giant nanobacterial reservoir in Africa: A significant fraction of a test group (40 HIV-infected mothers and 13 babies) was infected with nanobacteria. Concurrently, a multitude of 80-300 nm nanovesicles, apparently nanobacteria, were detected in the atmosphere of the Earth. Nanobacterial infections in HIV are possibly comparable to the twin epidemics HIV and tuberculosis. Models inspired by proteomics recommend methods to inactivate nanobacteria (and other slimeproducing bacteria) in the body. Keywords: nanobacteria • primordial proteins • slime • calcium phosphate • RNA • DNA • HIV
Introduction The functions of primordial proteins are investigated in an extended nanobacteria (NB) model. Previously, we have analyzed the survival modalities of NB in various environments, and could identify two complementary functions, adhesion and surface sealing, both securing the survival of NB in the extreme environmental conditions supposed to prevail in a primordial world. A third function, which can be related to a primordial scenario, is the effective collection, storage (and delivery) of the mineral constituents of the apatite shell protecting the NB. The catalog can be extended by the collection of nutrients. Laboratory experiments provided manifest evidence for at least one function, which appears to be useless in a primordial world, but plays an essential role in securing the survival of NB in the body of mammals: slime synthesis in response to external stimuli, e.g., alterations in the physiological milieu (i.e., blood). Remarkably, there is a complex interplay between these functions. For example, when the slime layer enveloping the NB dries, its bioadhesive capacity must decrease, consequently both functions adhesion to surfaces and collections of the mineral constituents must become inactive. An environment where these functions are superfluous is the dry atmosphere, external to clouds. There, surface sealing is practically the sole function temporarily inactive NB need to survive. To recognize the reciprocally exclusive functions of the proteins synthesized by NB is of paramount importance in the design of therapies allowing for inactivation of NB in the body. By interpreting NB as origin of life systems, the concept that the proteins synthesized by them are primordial proteins is evident. This position indicates, however, that the foremost biological functions of primordial proteins defined in NB are possibly conserved and * To whom correspondence should be addressed. E-mail: samoan@ gmx.net. † Present address: U.S. Department of Agriculture, Western Regional Research Center, 800 Buchanan, Albany, CA 94710. 10.1021/pr049795p CCC: $30.25
2005 American Chemical Society
still present in more advanced forms of bacteria. Microorganisms employ an arsenal of strategies to cause disease. The majority start by creating a connection with cells using simple techniques. Depending on the nature of the microorganism, the attachment could be specific or unspecific. Proteins are the principal actors in this field. To understand the versatility of their functions in the NB model is a motivating challenge in proteomics, providing a map for the systematic discovery of therapeutic responses to a relatively constricted spectrum of highly efficient primordial survival techniques, probably still active programs, and still successfully employed by many bacteria. The theoretical analysis of the functional properties of the primordial proteins in NB, applied in environments as different as the clouds and the blood circulation, has led to a number of predictions. The most prominent predictions included the prevalence of NB in HIV-infected patients, and in the terrestrial atmosphere. The expectation of a link between these occurrences propelled novel discoveries. Here, we proceed with the functional analysis of early proteins and extend the range of our model. For clarity and motivation, we summarize first some recent findings. Our model is based on a set of elementary premises required by a hypothetical primal biosystem for growth and replication in interplay with primordially available energy forms, i.e., solar light and water. The leading hypothesis is that in a primordial world, earliest forms of life must have possessed mechanisms to access and exploit the most abundant forms of energy by the use of the simplest physical and chemical principles. How fruitful this conceptual approach can be, is demonstrated in the following.
Nanobacteria in HIV There is growing observational evidence based on animal models, clinical studies and in situ measurements that NB excreted from the body of humans can reach the atmosphere Journal of Proteome Research 2005, 4, 633-636
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letters of the Earth. NB have been now identified in a representative number of HIV-infected patients in South Africa,1 confirming our earlier prediction with regard to the prevalence of NB in HIV-infected patients.2 In view of the nearly 30 000 000 HIVinfected persons in sub-Saharan Africa, the possible presence of NB in them could have an unforeseen impact on the spread of NB worldwidesand correlated with it, a boost to severe diseases including heart disease,3-6 kidney stones,7-9 peripheral neuropathy,2 and presumably reduced bone mineral density levels both in HIV-infected patients10 and astronauts exposed to microgravity.11 NB possess a unique capacity to survive in various milieus.12 Their slime provides protection from desiccation (exposing one key function of the primordial proteins). Previously, we have established a model, explicitly anticipating the presence and survival of NB in the stratospheresto the best of our knowledge, the first prediction of the existence of airborne NB,13 and subsequently also concluded that NB could be present in and excreted from HIV-infected people.2 By modeling the survival-oriented responses in NB, provoked by changes in the blood and the terrestrial atmosphere, an analysis of the adhesive-sealing functions of the primordial proteins (possibly glycoproteins),14 allowed us to design therapeutic applications of lasers to reduce NB concentrations in the body. Validation of our speculative model come from the experimental side, in the form of a substantial number of 80-300 nm airborne nanovesicles, captured in the upper stratosphere, via balloon.14 The correlations between the observable parameters of NB identified in humans,5-9 on the one hand, and those of the airborne nanovesicles, on the other hand, make it at present impossible to assume that the latter are not NB. Notably, their predominant size (∼300 nm), coincidences with the maximum size of the NB isolated from the body of humans, being about twice the mean size of possibly fossil NB detected on meteorite ALH 84001 (e100 nm).15 The smaller size seems reasonable, in particular because of the nutritional limitations prevalent on Mars. On the basis of the normal size-scale of NB isolated from human and animal sources, this comparison supports the argument that the majority of the nanovesicles collected via balloon were lifted from the Earth. This, in turn, leaves room to the possibility that they were excreted from the body of humans (and possibly animals). From spray irrigation and wastewater, NB could reach the atmosphere by a variety of physical processes. As recently reported, NB-like structures have been identified in wastewater in Korea.16 From open wastewater basins, NB may be thrown into the atmosphere by wind and waves, in a process corresponding to that by which sea-salt is known to be carried into the atmosphere.17 Another source may originate from the decomposition of cadavers of infected domestic and wild animals, exposed to the very strong African sun. In this scenario, NB can rise up into the atmosphere with the hot air around fires, and get transported by horizontal winds over long distances, as solitary aerosols or by attaching themselves to small biomass particles. While there is only limited quantitative information on the detailed mechanism by which NB could reach the upper stratosphere, a number of observations suggest that this is indeed possible for nanoscale particles.18
Targeting Primordial Proteins to Inactivate Nanobacteria Restricted to HIV, the models predict that at least four conditions which are relatively common in HIV, and considerably aggravate the state of the patients (heart disease, kidney stones, peripheral neuropathy, and reduced bone mineral 634
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density levels) are caused by NB. On the basis that NB have been finally detected in HIV, and the assumption that they contribute to the realization of the four conditions mentioned above, it seems now plausible that light intensities of the order of the solar constant (∼1000 Wm-2), previously observed to prevent slime production and modulate the vitality level of NB in vitro,19 will also be instrumental in ameliorating a central part of the diseases accompanying HIV-infection. This approach seems logical: the light is targeting one factor, causal for four separate diseases. Thus, selective irradiation of the body of HIV-infected patients with laser light, using irradiation parameters found to be effective in the therapy of peripheral neuropathy,2 a frequent condition in HIV, should show a strong synergistic effectsa novel therapy promising to help to stabilize the compromised immune system in HIV-infected patients. Calcium and phosphate, constituents of apatite, are prerequisites for the growth of the mineral shells of the NB. Growth is induced by stress.4 In vivo, stressed NB can only grow on cost of the calcium and phosphate circulating in the blood. In patients simultaneously infected with HIV and NB, the competition for calcium and phosphate offers an explanation for the reduced bone mineral densities observed in HIV.20-22 Prior to their excretion into the urine, NB have to pass the kidneys, where they can contribute to the nucleation of kidney stones.23 It becomes clear that a reduction of the NB concentrations in the blood, must reduce the number of NB potentially passing the kidneys, and thereby also the chances for the formation of kidney stones nucleated by NB.
Versatility of Primordial Proteins The versatility of the primordial proteins is reflected by the environmental adaptation of NB. A precondition for a therapeutic advance in this little-explored field is thus a complete understanding of both the slime’s chemical constitution and its interaction with visible light. It is therefore crucial to obtain, as soon as possible, a precise analysis of the proteins contained in and coating NB. Importantly, the slime combines a pronounced spinnability with a homogeneous consistencystypical for short proteins. Several authors have hypothesized that the original proteins were much shorter than most present day proteins and that the larger proteins were later assembled from these units.24-26 Recently, two solitary globular structures with diameters around 170 and 200 nm, respectively, imaged by scanning electron microscopy (SEM), were linked to airborne NB.27 On the basis of the premise that NB possess fimbriae, filamentous structures in the SEM image were interpreted accordingly. Fimbriae, tubular structures with diameters on the nanoscale, are found as external projections on many prokaryotic cells (bacteria). Bacterial fimbriae and pili represent highly organized assemblies of proteins, and short protein rods,28 respectively. Fimbriae execute principally one function: adhesion. From a functional point of view, the question whether primordial biosystems were equipped with both adhesive modes fimbriae and slime, which they may have used jointly or alternatively, or only with one, is of fundamental interest in proteomics, and can even have therapeutic relevance. Interestingly, high-resolution transmission electron microscopy images of present day NB show slime, but no further external projections that could be interpreted as fimbriae. Such fragile nanoscale features rarely survive fossilization and so are not available to the paleontologist.29 In such situations, valuable
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Primordial Proteins and HIV
indications can be extracted from models based on first principles, e.g., regarding the functions of primordial proteins in their native setting. NB slime is probably best described as an unorganized multifunctional random distribution of proteins and constituents of the apatitespossibly a variable barrier, depending on its density and viscositysand could serve at least four biological functions, including adhesion, surface sealing, as well as collection and storage of the mineral components of the apatite and nutrients.14 This picture leads to the following question: since NB possess such a useful tool allowing them to survive in almost every milieu, would nature have implemented fimbriae in them, as an extra adhesive modality? Here, ample experimental evidence on the ultrastructure of NBscontradicting the fimbriae theorysgives us the opportunity to decide the question. It could be argued that the two globular structures are not NB. This example illustrates the usefulness of models in providing reasonable hints on characteristics of the primordial proteins by analyzing the most vital functions required for the survival of the protein generators, so on the slime synthesized by contemporary NB.
Global Scale Transport Cycle There is a clear increase in the number of studies documenting the pathogenic potential of the NBsisolated in humans on four continents. NB have been identified in patients in Europe, China, India, South Africa, and the U.S.A. Because of their manifest contribution to the pathology of a multitude of severe diseases, it appears to us important to address a probable relationship between their excretion from possibly millions of HIV-infected individuals, in particular in sub-Saharan Africa, and their presence in the terrestrial atmosphere. With the possibility of their revitalization in clouds,30 the presence of NB in the atmosphere implies a worldwide distribution of viable NB. This perspective should justify to check the routes by which a considerable part of presumably tons of NB excreted daily from human reservoirs, might find access into the atmosphere. In many undeveloped countries, unfiltered (or insufficiently filtered) human excreta are still used for agricultural sprayirrigation. In a worse case scenario, 30% of the water is likely to evaporate before it could reach the soil. Winds and ascending hot air can carry the NB contained in evaporating water droplets into the air. It is documented that much larger dust particles are transported by winds as far as from Africa to the Americas.31 Similarly, NB suspended in the atmosphere could be easily distributed by winds over extended geographical zones. A part of them may eventually return to the Earth as aerosols. From clouds, viable NB could be transported to the Earth by precipitation30sa transport cycle which deserves the attention of both researchers and governments. NB have been detected in some human and veterinary vaccines.32 However, their presence in the atmosphere, and the associated possibility of their natural incorporation seems to represent a contamination route which is exceeding by far whatever could stem from vaccines. NB concentrations in clouds are far too small for a substantial contribution to precipitation, however, NB are probably excellent cloud condensation nuclei.30 In the clouds, NB may form larger rain drops, prior to other aerosols, and even encourage slime-mediated attachments to other microorganisms and/or biomass particles, thus finding further modalities to reach the Earth within precipitation.30 Due the detection of NB in HIV-infected people, the probability for regionally elevated NB concentrations in human excreta and wastewater now seems realistic. A reliable model accounting
for the emission of NB into the atmosphere requires first a quantitative analysis of their presence in these resources. NB were included in a report on possible drinking water contaminants (Report of the CCL Classification Process Work Group to the National Drinking Water Advisory Council, May 19, 2004).
Indication for a Protein-Mediated Active Role of Nanobacteria in HIV By analyzing survival modalities of NB in space,13 we could identify several intrinsic functions of their slime. The versatile functions of the NB slime precisely define the most basic evolutionary requirements of the primordial proteins. NB seem to spontaneously respond to light stimuli in their environment. Variations in light intensity, time of exposure, and air humidity are presumably signals, switching between the complementary functions “sealing” and “sticking”.10 A better understanding of the proper role of the primordial proteins participating in these evolutionary programmed processes could be exploited therapeutically, e.g., by creating adverse situations, blocking or activating the ancient program of the NB (and possibly also of many other bacteria). HIV-infection presents massive physiological changes to the NB circulating in the blood. In vitro experiments showed that cultured NB respond to changes in their environment by producing slime, a means not only to adhere to surfaces to prevent elimination from the body, but also to acquire additional amounts of calcium and phosphate, required to enforce the protective mineral shell.4 The instant occurrence of localized calcium phosphate gradients in the circulation, promoted by stimuli provoking the synthesis of the protein forming the slime envelope of the NB, could possibly have an extensive impact on processes controlling the uptake of nucleic acids by cells. Dissolved constituents of the apatite, transiently stored in the slime of NB attached to cells, or even internalized by NB entering cells,7 could represent such gradients. They may influence equivalently physical and biochemical processes, both on the surface and in the interior of cells. In laboratory experiments with human eukaryotic cells (e.g., lymphocytes), calcium phosphate was found to be a key player in mediating RNA (DNA) uptake.33-35 Several interaction modes between elements of the triple system virus-cell-NB appear possible. The following scenario deserves attention: Calcium phosphate, present in the slime of a stressed NB internalized in an HIV-infected lymphocyte, may facilitate the entrance of viral DNA into the cell’s nucleus.
Conclusions We have possibly identified a relevant source of airborne NB, as well as a further element in the pathology of HIV. In our previous work, the implication of NB in HIV was understood as a passive phenomenon that can be summarized as follows: due to various massive physiological changes in the blood, concomitant with a compromised immune system, NB are stimulated to grow and replicate. By secreting slime, they can form colonies and/or adhere to cells, merely acting as opportunistic infectious agents. In the active role, induced in a virtually similar way, the process of collection of calcium phosphate in the slime layer could represent the beginning of a cascade of synergistic events, possibly implicated in a rapid progress of the HIV-infectionsin patients with high levels of NB in the blood.
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