Potentially conflicting selective forces that shape the vls antigenic variation system in Borrelia burgdorferi
Introduction
Changes in environmental conditions are a common source of natural selection driving adaptation in natural populations. While adaptation to predictable or cyclic environmental changes has been the focus of numerous studies (for example, Cronin and Schneider, 1990, Erwin, 2009, Merila, 2012), adaptation to unpredictable and rapidly changing environments is less well characterized. However, unpredictable and rapidly changing environments are common and can result in lethal selection pressures, such as the adaptive responses of vertebrate immune systems that shape the evolutionary dynamics of pathogen populations. The persistence of pathogens within vertebrate hosts in the face of the potentially lethal environmental conditions imposed by the immune system is a primary constituent of the evolutionary fitness of many microbial pathogens (Brunham et al., 1993, Combes, 1997, Deitsch et al., 1997, Schmid-Hempel, 2009). These strong selective pressure imposed by the immune response has resulted in antigenic variation mechanisms evolved to cope with this rapidly changing environment (Brunham et al., 1993, Deitsch et al., 1997, Frank, 2002, Moxon et al., 1994, Schmid-Hempel, 2009).
Antigenic variation systems alter surface antigens of pathogens, giving rise to subpopulations of pathogens with distinct antigenic variants that are not recognized by antibodies targeting previously detected antigens (van der Woude and Baumler, 2004). Evading the antibody response permits longer residence times of the pathogens within the host, thus increasing opportunities for transmission to naïve hosts (Deitsch et al., 1997, Moxon et al., 1994). Antigenic variation systems that more efficiently alter the antigenic surface of the pathogen are likely to be selectively advantageous as they promote greater residence time within hosts and transmission to naïve hosts, both of which are primary components of pathogen fitness.
Many pathogens utilize antigenic variation systems that alter the genetic sequence in the expression site of the antigenic protein (Deitsch et al., 2009; van der Woude and Baumler, 2004). One common molecular mechanism involves recombining gene fragments from unexpressed, paralogous ‘cassettes’ into an expression site, thereby altering the sequence of the expressed antigen. In these types of recombination-based antigenic variation systems, which are common in several bacterial genera (Hagblom et al., 1985, Noormohammadi et al., 2000, Zhang et al., 1997), the ability to alter the sequence of the expressed antigen is correlated with the amount of diversity among the unexpressed cassettes. Thus, natural selection should favor ever greater diversity among unexpressed cassettes to promote ever greater divergence among expressed antigens (Graves et al., 2013, Lipsitch and O’Hagan, 2007). However, the extent of the divergence among cassettes can be constrained by other features of the system (Haydon and Woolhouse, 1998). Here, we use the well-characterized vls antigenic variation system in the Lyme disease bacterium, Borrelia burgdorferi, as a model system to investigate the interactions between selection favoring greater antigenic divergence and other potential constraints on antigenic variation systems.
B. burgdorferi requires continuous alteration of the highly-expressed VlsE antigen for long-term survival within hosts (Bankhead and Chaconas, 2007, Bykowski et al., 2006, Labandeira-Rey and Skare, 2001, McDowell et al., 2002, Purser and Norris, 2000, Rogovskyy and Bankhead, 2013, Zhang et al., 1997). A fragment of an unexpressed vls cassette can be introduced into the vlsE expression site through nonreciprocal recombination, thus changing, adding, or removing nucleotides in sequence of the expression site resulting in the expression of a divergent VlsE antigen. However, altering the sequence in the expression site could potentially reduce the ability to translate a functional protein – by introducing stop codons or frameshift mutations – or reduce translational efficiency and accuracy – by introducing non-preferred codons (Coutte et al., 2009, Hershberg and Petrov, 2008). Little is currently known about how selection on translational ability or efficiency constrains the nucleotide identities at the polymorphic sites, positions of the polymorphic sites and positions of the insertion/deletion mutations.
Here we evaluated the effects of the identity of nucleotides at polymorphic sites, positions of the polymorphic sites, and position of insertion/deletion mutations in the unexpressed cassettes on the divergence among antigenic variants as well as their translational ability and translational efficiency. We ask if the organization of polymorphic sites and insertion/deletion mutations in the unexpressed cassettes of multiple natural strains results in the greatest possible antigenic divergence, translational ability, and translational efficiency in the VlsE variants. We used in silico simulation models to test if perturbing the observed polymorphic sites leads to a decrease in antigenic divergence, translational ability and translational efficiency.
Section snippets
Sequence analysis of vlsE and the unexpressed cassettes
The sequences of the unexpressed cassettes from six strains of B. burgdorferi sensu stricto and one Borrelia afzelii strain were used to investigate how diversifying selection and translational selection constrain identities and locations of polymorphism among the unexpressed cassettes (Table 1). Each of the unexpressed cassettes within each strain was aligned using ClustalW (Larkin et al., 2007) with default parameters. The unexpressed vls cassettes from all strains have six or seven variable
Effect of altering the nucleotide identities observed in the polymorphic sites
The nucleotide identities observed in polymorphic sites of the unexpressed cassettes of natural strains result in the greatest antigenic divergence, translational ability, and translational efficiency in VlsE variants. Changing nucleotides at the sites that are naturally polymorphic (δNuc) among the unexpressed cassettes dramatically reduced the antigenic divergence among VlsE variants as well as the translational ability and translational efficiency of the variants (Fig. 2). The reduction in
Discussion
Antigenic variation systems experience strong selection to evade the rapidly changing and lethal host immune environment. Thus, there is a premium for ever greater antigenic divergence among protein variants generated by antigenic variation systems. However, divergence among antigenic variants as well as the organization of the antigenic variation system as a whole can be constrained by selection for basic molecular functions such as translational ability or translational efficiency. The
Acknowledgments
We thank Steven Norris for assistance with the project, and Junhyong Kim, Paul Sniegowski, Rahul Kohli and Timothy Linksvayer for helpful suggestions.
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