Sequence-Based Classification of Select Agents A Brighter Line (2010) / Chapter Skim
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2 Challenges of Predicting Pathogenicity from Sequence
2 Challenges of Predicting Pathogenicity from Sequence
Pages 37-72
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From page 37... ...
pathogens, that is, microorganisms capable of causing disease. Most Select Agents are not typical of the common pathogenic microorganisms seen in human or animal medicine, or in agricultural practice.
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From page 38... ...
reported that 3,606 bacterial genomes were being sequenced and that complete DNA se quences of at least 712 distinct bacterial strains were in the public domain. The completed sequences include all the bacterial Select Agents and most common pathogens of humans, animals, and plants.
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We have also begun to understand how pathogens got to where they are and, we know to some extent what to look for if we are trying to design a pathogenic microorganism. The objective of a "predictive oversight" system would be to forecast with a high degree of certainty the pathogenic potential of sequences of Single or small numbers of genes related to Select Agent toxins.
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From page 40... ...
2 It is this last level of complexity that gives rise to the key biological attributes of pathogenicity and transmissibility, factors that contribute to the criteria that form the basis of inclusion of an organism on the Select Agent list. Predicting pathogenicity or transmissibility of a microorganism requires a detailed understanding of multiple attributes of both the pathogen and its host.
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, whereas the portion of the enzyme that recognizes the specific DNA sequence cleavage site might evolve rapidly (related enzymes cut different DNA sequences)
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This is not a perfect means of prediction, how ever, inasmuch as the 1918 influenza virus, which was associated with 50 million deaths worldwide, has a cleavage site that appears from sequence analysis to be associated with low pathogenic potential (Box 2.1)
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Yet, the correlation of high pathogenicity and trypsin independent cleavage of HA is not perfect; the 1918 influenza virus, which was associated with 50 million deaths worldwide, has a cleavage site that appears from sequence analysis to be of the low pathogenic. Data Bank contains over 60,000; http://www.rcsb.org/pdb)
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SEQUENCE-BASED CLASSIFICATION FOR SELECT AGENTS BOX 2.2 Critical Assessment of Protein Structure Prediction (CASP) Competition Last year's Critical Assessment of protein Structure Prediction (CASP8)
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From page 45... ...
Gene Regulation If an organism's virulence depends on specialized gene products, it must be able to use them when they are needed but not squander its metabolic energy in producing them aimlessly or risk having them detected and prematurely neutralized by host defenses. Consequently, regulating the expression of virulence factors is an additional essential complication of a pathogenic microorganism's life.
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From page 46... ...
Moreover, many microorganisms produce toxins that are regulated by iron in such a way that low iron concentrations trigger toxin biosynthesis. Reversible regulation of the expression of virulence genes by temperature is common to many pathogens.
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Thus, the inherent pathogenicity of a microorganism can be greatly altered through regulation of virulence genes. It is extremely difficult to predict how even a single nucleotide change will affect regulation and thereby alter patho genesis or the viability of the microorganism.
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Many of these microorganisms are among the most deadly for humans and are dispro portionately represented on the Select Agent list of potential bioterrorism agents. Generally speaking, these microorganisms are distinguished from human-specific pathogens in not being directly or readily transmissible from one human host to another.
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From page 49... ...
Camelpox and taterapox viruses are the closest orthopoxvirus relatives of variola virus and have similar number of virulence genes, but nei ther virus causes human disease. Cowpox virus has a larger genome than does variola virus and encodes a greater number of virulence genes, but it causes only a localized lesion in humans and was used by Jenner in the late 1700s to vaccinate against variola virus.
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. In the case of bacteria, the picture is a little clearer inasmuch as bacterial pathogens usually have virulence genes that are not present in their non-pathogenic rela tives, and this distribution suggests that bacteria evolve to become pathogens by acquiring virulence determinants.
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cereus, is not always clear in a sequence homology context, whereas the sequence information makes good sense in a biological context. For a more in-depth discussion of pathogenic mechanisms and virulence genes with examples of Select Agents, see Appendix I
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SEQUENCE-BASED CLASSIFICATION FOR SELECT AGENTS FIGURE 2.1 The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Outer circle, predicted 1 Figure 2- coding regions on the plus strand colour-coded by role categories.
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For present purposes, BOX 2.5 Acquisition of Virulence Factors -- Horizontal Gene Transfer Horizontal (or lateral) gene transfer is any transfer, exchange or acquisition of genetic material that differs from the normal mode of transmission from parents to offspring (vertical transmission)
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If we trace the kinds of genes that appear to be peculiar to pathogens, we see that similar kinds of genes encode toxins or invasins found in organisms that infect plants, people and organisms in between. To some extent, we find that virulence genes evolved to prevent predation.
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But it consti tutes the most practical means of identifying pathogenic potential today, and it probably will lead to a much greater predictive capacity in the future. The Evolution of Bacterial Host Specificity If the acquisition of genes by horizontal gene transfer seems to be a driv ing force for bacterial pathogenicity, one might assume that gene loss plays only a minor role in pathogenicity.
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have a number of pathogenicity islands that were acquired by horizontal gene transfer. The Salmonella group of pathogens is notable for its division into specific types that have a preference for a particular host (animal, bird or reptile)
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As the pattern of gene gain through horizontal gene transfer and subsequent gene loss through adaptation has been analyzed, it appears that gene loss may be a mechanism of targeting the invading pathogen preferentially to particu lar tissues or host cells and avoiding the potential stimulation of non-specific inflammation. For example, in both Salmonella and Yersinia, gene loss may be involved in the adaptation from a gastrointestinal to a systemic "lifestyle." Similarly, genomic analysis seems to refute the popular belief that M
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From page 58... ...
As part of this process the new spe • cies acquires some barrier to genetic exchange with the parent species. expansion of paralogous gene families and acquisition -- by horizontal gene transfer -- of numerous genes from early eukaryotic hosts, other viruses and, rarely, bacteria.
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From page 59... ...
The evolution of two distinct poxvirus pathogens that cause dissimilar human disease from a common ancestor through gene loss suggests that there can be multiple genetic pathways to becoming a pathogen for the same host. Furthermore, inasmuch as the genomes of molluscum contagiosum and variola viruses are distantly related with 57 percent nucleotide identity (on the basis of DNA polymerase)
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. Monkeypox and variola viruses together have at least 33 orthopoxvirus virulence genes where a function has been determined for the actual gene or for an orthologue in another orthopoxvirus species.
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. A stable microbe-host interaction results in survival of both the microorganism and host on a population basis (for example, variola virus, polio virus or M
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The majority of the human pathogens found on the Select Agent list cause dead-end interactions. The outcome of a microbe-host encounter is based on interactions at the molecular and cellular level that take place over time.
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that was the precursor to the SARS-CoV epidemic, and the first complete synthesis of a 582 kb artificial bacterial genome. Most recently, a synthetic bacterial genome has been "booted"6 into an autonomous life-form, so the artificial bacterial genomes are self-perpetuating (Gibson, Glass et al.
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From page 64... ...
Another concern is the deliberate misuse of the technology to design and construct new pathogens, either by engineering in components that resist current vaccine or therapeutic interventions, or by altering pathogenesis by blending in virulence genes from alterative pathogens, or by the de noo design of new pathogens. Understanding of the complex genetic and protein networks that regulate replication and disease is substantially lim
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. There are proven strategies exist for reconstituting most of the viral Select Agent and category A-C biodefense pathogens from full-length DNA genomes; however, infectious clones have not been constructed for many Select Agent viruses on these lists.
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As noted throughout this chapter, the list of "virulence genes" that have defined biological properties is growing at a considerable rate, and this fuels concerns that virulence is a readily malleable trait. Limited research has focused on the potential of synthetic genome design to enhance viral pathogenesis.
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in the BMBL. Of these, some are designated Select Agents, and a few are prioritized under the DHS Bioterrorism Risk Assessment.
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Designing an infectious viral genome de noo by sequence requires the accurate prediction of protein structure and function, the design of protein-protein interactions and protein machines, all of which must produce progeny virions efficiently in an order of magnitude more complex host cell. If we cannot predict protein structure and function on the basis of sequences with any accuracy, how can we design and synthesize novel viruses that will replicate, regardless of their disease potential?
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From page 69... ...
WHAT CAN CURRENTLY BE PREDICTED FROM SEQUENCE ABOUT THE IDENTIFICATION OF PATHOGENIC MICROORGANISMS, INCLUDING SELECT AGENTS? It is abundantly clear that the use of sequence alone to predict a naturally occurring or synthetic pathogenic microorganism accurately is infeasible -- se quence cannot provide biological context.
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From page 70... ...
Just as we can tell from the sequence of "core" genes that we are deal ing with a bacterium of the genus Yersinia or a poxvirus, we can accurately say whether there is a precise sequence of a known virulence gene associated with the plague bacillus or with smallpox. It might be difficult to identify the precise origin of a microbial sequence to a specific Select Agent, but it is surely less difficult to say with some accuracy that a particular sequence is related to a known virulence determinant and to a class of virulence genes with known function.
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If the sample were submit ted to a DNA synthesis company, there would be cause to learn more about its source and about the reason the DNA was being synthesized.8 There is no fool-proof method for predicting whether a sequence will have the biological properties of a Select Agent (such as pathogenicity) , but the common sense use of the considerable amount of sequence data we now have, combined with advances in understanding of microbe-host interactions, does provide for a mechanism that is practical and sufficiently flexible to provide guidance about the potential biological consequences of a DNA sequence from nature or from the biochemist's bench.
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Moreover, we present a "yellow flag" biosafety system; this approach is not regulatory and therefore could provide sequence information relevant to biosecurity in a more dynamic and timely manner than the Select Agent Regulations.
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