Primordial Proteins and HIVsPart II Andrei P. Sommer* Central Institute of Biomedical Engineering, University of Ulm, 89081 Ulm, Germany Received March 15, 2005
Nanobacteria are suspected to be responsible for a number of diseases, i.e., kidney stones, heart disease, ovarian cancer, peripheral neuropathy, and reduced bone mineral density. Being protected by a mineral shell consisting of apatite, the nanovesicles can enter eukaryotic cells. Depending on the host’s stress level, nanobacteria may carry a substantial layer of a protein based slime, instrumental in collecting calcium phosphate from the environment. Calcium phosphate is known to mediate the uptake of nucleic acids by eukaryotic cells. Surprisingly, a pathogenic effect of nanobacteria in HIV can be derived primarily from the trafficking of calcium phosphate in HIV infected cells, performed by primordial proteins. The inescapable conclusion is that nanobacteria could promote genetic diversity in HIV. Keywords: nanobacteria • primordial proteins • calcium phosphate • transfection • RNA • DNA • HIV
Introduction Since the concept of nanobacteria (NB) has been introduced to the wide public in 1998, the subject has become increasingly provocative, dividing the scientific community into two partiess believers and nonbelievers. The constant ambivalency of the subject is best reflected by three articles,1-3 published between 1999 and 2004, and has basically to do with a series of experimental-clinical papers in which the authors reported on the identification of nucleic acids (RNA or DNA) in NB.4-7 Since NB are very small (60-300 nm), identification of nucleic acids in them is admittedly a scientifically attractive challenge. Ironically, critics of the NB employ the same size-argument to drive the whole NB concept ad absurdum. These debates have finally contributed to a shift in the positions, with an apparent territorial gain for those rejecting NB as a form of life, and brought the pioneers who continued to stick to the nucleic acid paradigm, supporting it with more or less convincing experiments, into the focus of massive critics. One unfortunate consequence of these polarized battles was that opponents of the nucleic acid theory were ready to disqualify the entire NB concept.8 On a longer term, a cautious open attitude may stimulate the reorientation of both sides with a “return to first principles” (recommended by Machiavelli). Results of clinical studies and laboratory experiments performed by independent groups suggest, however, that it might be worth to consider the NB problem from another side, to strictly focus on what can be regarded as ascertained and to postpone for some time the direct confrontation with the critical question: are NB alive or not? Instead, we concentrate here on three intrinsic properties of NB: 1. protection by a permeable mineral shell consisting of apatite,4 2. synthesis of slime and growth in response to environmental stress (equivalent to biomechanical and/or physiological variations in the blood),9 and 3. susceptibility to * To whom correspondence should be addressed. E-mail: samoan@ gmx.net.
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visible light of intensities and doses applied at biostimulatory standards.10 The latter was shown to elevate the vitality level of a large body of (stressed) mammalian cells, in vitro and in vivo,11 and observed to block the production of slime in cultured NB.12 Interestingly, the pathogenic nature of NB can be traced back solely to the synergistic interplay of the first two properties.13-15 Conversely, therapies to reduce concentrations of NB in the body could be derived from the last two properties.16-18 This becomes clear by realizing that without the protein based slime, the bioadhesive capacity of NB must decrease.15 Consequently, the probability of a partial elimination of NB via urine must increase. Excretion of NB in urine has been reported in animals.19 Actually, the response of NB to photobiostimulation,10,12 observed in two labs, is an irrefutable indication that NB have indeed something in common with conventional biosystems.
Primordial ProteinssCollection and Carrier Proteins Considering the unique chemical composition, extensive survivability, and protective architecture suited to harvest and exploit solar irradiation, we have argued that the proteins synthesized by NB could be primordial proteins.20 In a recent paper, analyzing the functions of primordial proteins in the NB model, we have formulated a catalog of the properties that primordial proteins must have had in order to secure the survival of NB in extreme environments. The most prominent functions were adhesion and surface sealing, as well as collection and storage of the mineral components of the apatite and nutrients.21 Collection of the mineral components of the apatite follows directly from the fact that NB growsboth in culture and in vivoson the cost of the calcium and phosphate present in these milieus. However, due to a slime envelope, acting presumably also as a chemical barrier, before integration into the mineral shell, the collected components are transiently stored in the slime.22 Growth implies the diffusion of the collected material across the slime film. The picture emerging 10.1021/pr050064e CCC: $30.25
2005 American Chemical Society
letters
Primordial Proteins and HIVsPart II
Challenges for Clinical Studies and Laboratory Experiments
Figure 1. Artist’s view of double-infected human lymphocyte (616 µm). Two NB (60-300 nm) have already passed the cytoplasm (C) and adhere to the nuclear membrane. Primordial proteins coating NB and enriched with calcium phosphate should mediate the uptake of viral DNA by the nucleus (N). Due to the predominantly short proteins, slime coating NB will have a pronounced fluidity.22 This indicates a spread of the slime on the nuclear membrane, exceeding the dimensions of the NB, thereby increasing the likelihood that viral DNA, prior to traversing the membrane, could contact fields with elevated levels of calcium phosphate. The spread of the slime could be limited by the residence time of the NB at the membrane, which may even cross the nuclear membrane, creating channels facilitating the entrance of viral DNA.
from this process is a calcium phosphate gradient, perpendicular to the slime barrier. Its presence in the blood circulation may have an enormous impact on the interaction between the HIV virus and immune defense. Calcium phosphate is known to promote the uptake of both RNA and DNA by eukaryotic cells.23-25 The mechanism of the uptake is still not precisely understood: calcium phosphate might affect transfer processes across the cell membrane, nuclear membrane, or processes in the cytoplasm. Nucleic acid transfection was discovered with calcium phosphate as a natural mediator and DNA, and published in 1973.26 However, calcium phosphate was soon ousted from its initial position in genetic engineering by other, more effective additives. A renaissance may come from the identification of NB in HIV-infected patients,27 in conjunction with earlier reports on the internalization of NB by eukaryotic cells.4,19 The possibility that calcium phosphate, stored in the slime film coating NB, might be involved in the transfection of lymphocytes with viral nucleic acids seems realistic.22 Slime could have been synthesized external to cells, or even upon entry. It should be explicitly pointed out that without the collection, storage, and carrier function of the primordial proteins, calcium phosphate would have only a small chance to infiltrate eukaryotic cells in vivo. Clearly, the implication of NB in viral nucleic acid transfection does not depend to any extent on their biological classificationsonly the prevalence of calcium phosphate in the protein film coating the nanovesicles is relevant. With NB co-infection, the intensity (number of infected lymphocytes) and the kinetics (progress) of the HIVinfection could simultaneously increase, resulting in an extensively destructive effect. A simplistic representation of the principle of one possible NB-HIV-double infection scenario is illustrated in Figure 1.
The principal elements of the current tentative model, established in our preliminary work22sthe apatite shell and the slime envelopesare both well-documented in the published literature.4,21 Its verification needs an approach from two different sidesslaboratory studies designed to check for the presence of NB in HIV-attacked lymphocytes, and clinical studies comparing the temporal development of the HIVinfection in geographical zones with high and low concentrations of NB in the blood, respectively. It is expected that elevated NB concentrations are coincident with elevated levels of opportunistic infectious agentsstherefore, even the potential outcome of a significant correlation between a progress in the HIV-infection and co-infection with NB will not automatically signify a real correlation between the two events. This requires validation from laboratory experiments: detection of NB with their slime envelope in HIV-infected lymphocytes. Co-localization of viral nucleic acids and slime enveloping NB as small as 60 nm, internalized in cells reaching several microns, is in no way trivial and demands for high-resolution imaging methods operating in ambient conditions or in aqueous liquids, e.g., environmental scanning electron microscopy (E-SEM), environmental transmission electron microscopy (E-TEM), or nearfield optical analysis (NOA). NOA is performed via a near-field scanning optical microscope (NSOM) operating in the reflection mode and equipped with nanostructured titanium substrates.28 The performance of E-TEM for such proposes was recently demonstrated by visualizing nanoparticles at the interface of two different liquids.29 Remarkably, the NB problem is not only a serious challenge to the clinical and experimental-microbiological side but also to nanotechnology, and here especially to models. While it is known that with suitable assistance, DNA and RNA can enter and escape from a cell, the question whether nucleic acids possibly present in NB could ever escape from intact mineral nanocapsules cannot be answered without complicated experiments. The central momentum in them would be the clear proof that nucleic acids possibly stemming from mineralized NB (and detected in a medium containing such NB) are definitely from NB, and not from other biological sources. Solving this also evolutionary relevant problem, involves information on the transport of nucleic acids through nanoscale apertures mimicking the pores present in the mineral shell of NB. Such models exist,30 were tested experimentally and suggest themselves to estimate the chances for the release of RNA (probably contained in NB) from NB. This would require reasonable data on the maximum diameter of the pores in the mineral shell of NB. Because of the virtually rough surface topography of the shell, reliable data could be obtained by the use of adequate tracer liquids, and subsequent fragmentation of the NB capsule. Due to the reported presence of NB in HIV-infected patients in Africa, NB deserve our attention. Clarification of their potentially active role in HIV-infection (and possibly in other diseases induced by uptake of foreign nucleic acids by cells) now seems important. In addition to the aforementioned laboratory experiments and clinical surveys focusing on characteristics of the HIV-infection for different geographical zones, mathematical simulations accounting for demographic determinants may provide valuable complementary data allowing us to compare and interpret local factors. Geographical coincidence between high HIV infection rates and traditional use of human excreta for agricultural irrigation suggests localizable environmental pools containing viable NB, Journal of Proteome Research • Vol. 4, No. 3, 2005 1023
letters with an absolute maximum in sub Saharan Africa. Changes in immunocompetent cells prevailing during the primary HIV infection may activate (permit?) NB replication. Via HIV DNA transfection,22 NB could accelerate the viral turnover in an HIV infected individual. Rapid turnover is regarded as one factor responsible for the genetic diversity of the HIV-1 virus31shaving its maximum in Africa. By facilitating the transfer of HIV DNA across the nuclear membrane of a cell harboring different viral strains of HIV, the considered transfection processes could increase the odds of its simultaneous infection with two different proviruses (precondition for recombination events leading to genetic diversity in HIV). Finally, NB entering HIV infected lymphocytes during the asymptotic phase of the HIV infection could represent stimuli affecting the long term stability of the 100:10:1 ratios estimated between the latently infected lymphocytes containing HIV DNA, those with integrated HIV DNA, and those producing virions, respectively.32
Conclusions NB previously injected in a rabbit and then isolated from the urine of the animal were reported to invade the nuclei of 3T6 cells in vitro. Moreover, NB from the original culture were reported not to invade effectively, but to adhere to 3T6 cells.19 Presumably, the adherence of NB from the original culture to 3T6 cells was a process mediated by slime, whereas those invading the nuclei carried less slime. On the basis of the assumption that viable NB are excreted from a considerable fraction of the 30 000 000 HIV-infected people in sub Saharan Africa, we arrive at the conclusion that on passing the high perfusion zone of the kidneys, bath in the bladder and excretion in the urine, the bioadhesive slime on NB, apparently based on a glycoprotein, is washed awaysat least partially. This could mean that NB excreted from HIV-infected patients (probably the largest existing NB reservoir) possess a highly infective potential. With traces of calcium phosphate in a minimally thin protein film coating the apatite shell, re-incorporation of NB could enhance mortality in HIV by entry into lymphocytes.22 Laboratory experiments allowing to evaluate the effects of calcium phosphate imported by cultured and urine-excreted (washed) NB into HIV-infected lymphocytes are necessary to verify the infection-transfection mechanisms discussed in this model. The possibility of a profound epidemiological implication should motivate a global scan for viable NB loadssin human excreta used in agricultural irrigation and fertilizers, and the environment.
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Acknowledgment. I dedicate this work to Dr. Attila Pavla´th, on the occasion of his 75th birthday. References (1) Abbott, A. Nature 1999, 401, 105. (2) Cisar, J. O.; Xu, D.-Q.; Thompson, J.; Swaim, W.; Hu, L.; Kopecko, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11511. (3) Hogan, J. New Scientist 2004, May 19. (4) Kajander, E. O.; Ciftcioglu, N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8274. (5) Miller, V. M. et al. Am. J. Physiol. Heart. Circ. Physiol. 2004, 287, H1115. (6) Khullar, M. et al. Urol. Res. 2004, 32, 190. (7) Hudelist, G. et al. Histopathology 2004, 45, 633. (8) Godfarb, D. S. Nephron Physiol. 2004, 98, 48. (9) Sommer, A. P.; Pretorius, A. M.; Kajander, E. O.; Oron, U. Cryst. Growth Des. 2004, 4, 45. (10) Sommer, A. P.; Hassinen, H. I.; Kajander, E. O. J. Clin. Laser Med. Surg. 2002, 20, 241. (11) Sommer, A. P.; Oron, U.; Kajander, E. O.; Mester, A. R. J. Proteome Res. 2002, 1, 475. (12) Sommer, A. P. et al. J. Clin. Laser Med. Surg. 2003, 21, 231. (13) Sommer, A. P. J. Proteome Res. 2004, 3, 667. (14) Sommer, A. P. J. Proteome Res. 2004, 3, 1086. (15) Sommer, A. P.; Cehreli, M.; Akca, K.; Sirin, T.; Piskin, E. Cryst. Growth Des. 2005, 5, 21. (16) Sommer, A. P. J. Proteome Res. 2003, 2, 665. (17) Sommer, A. P. J. Proteome Res. 2004, 3, 670. (18) Sommer, A. P.; Wickramasinghe, N. C. Int. J. Antimicrob. Agents 2004, 24, 548. (19) Akerman, K. K. et al. Proc. SPIE Int. Soc. Opt. Eng. 1997, 3111, 436. (20) Sommer, A. P.; Miyake, N.; Wickramasinghe, N. C.; Narlikar J. V.; Al-Mufti, S. J. Proteome Res. 2004, 3, 1296. (21) Sommer, A. P.; Wickramasinghe, N. C. J. Proteome Res. 2005, 4, 180. (22) Sommer, A. P.; Pavla´th, A. E. J. Proteome Res. 2005, 4, 633-636. (23) Kleinschmidt, A. M.; Pederson, T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1283. (24) De Lucca, F. L.; Sawan, F. M.; Watanabe, M. A.; de Souza, L. R. Mol. Cell. Biochem. 2001, 228, 9. (25) Sabelnikov, A. G. Prog. Biophys. Mol. Biol. 1994, 62, 119. (26) Graham, F. L.; van der Eb, A. J. Virology 1973, 52, 456. (27) Pretorius, A. M.; Sommer, A.P.; Aho, K. M.; Kajander, E. O. HIV Med. 2004, 5, 391. (28) Sommer, A. P.; Franke, R. P. J. Proteome Res. 2002, 1, 111. (29) Dai, L. L.; Sharma, R.; Wu, C.-y. Langmuir 2005, in print. (30) Storm, A. J.; Storm, C.; Chen, J.; Zandbergen, H.; Joanny, J.-F.; Dekker, C. Nano Lett. 2005, in print. (31) Lal, R. B.; Chakrabarti, S.; Yang, C. Indian J. Med. Res. 2005, 121, 287. (32) Wain-Hobson, S. Nature 1997, 387, 123.
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