Identification of core DNA elements that target somatic hypermutation.

Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 3' element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.

Evolution of polyamine resistance in Staphylococcus aureus through modulation of potassium transport.

Microbes must adapt to diverse biotic and abiotic factors encountered in host environments. Polyamines are an abundant class of aliphatic molecules that play essential roles in fundamental cellular processes across the tree of life. Surprisingly, the bacterial pathogen Staphylococcus aureus is highly sensitive to polyamines encountered during infection, and acquisition of a polyamine resistance locus has been implicated in spread of the prominent USA300 methicillin-resistant S. aureus lineage. At present, alternative pathways of polyamine resistance in staphylococci are largely unknown. Here we applied experimental evolution to identify novel mechanisms and consequences of S. aureus adaption when exposed to increasing concentrations of the polyamine spermine. Evolved populations of S. aureus exhibited striking evidence of parallel adaptation, accumulating independent mutations in the potassium transporter genes ktrA and ktrD. Mutations in either ktrA or ktrD are sufficient to confer polyamine resistance and function in an additive manner. Moreover, we find that ktr mutations provide increased resistance to multiple classes of unrelated cationic antibiotics, suggesting a common mechanism of resistance. Consistent with this hypothesis, ktr mutants exhibit alterations in cell surface charge indicative of reduced affinity and uptake of cationic molecules. Finally, we observe that laboratory-evolved ktr mutations are also present in diverse natural S. aureus isolates, suggesting these mutations may contribute to antimicrobial resistance during human infections. Collectively this study identifies a new role for potassium transport in S. aureus polyamine resistance with consequences for susceptibility to both host-derived and clinically-used antimicrobials.

Dynamic Evolution of Bacterial Ligand Recognition by Formyl Peptide Receptors.

The detection of invasive pathogens is critical for host immune defense. Cell surface receptors play a key role in the recognition of diverse microbe-associated molecules, triggering leukocyte recruitment, phagocytosis, release of antimicrobial compounds, and cytokine production. The intense evolutionary forces acting on innate immune receptor genes have contributed to their rapid diversification across plants and animals. However, the functional consequences of immune receptor divergence are often unclear. Formyl peptide receptors (FPRs) comprise a family of animal G protein-coupled receptors which are activated in response to a variety of ligands including formylated bacterial peptides, pathogen virulence factors, and host-derived antimicrobial peptides. FPR activation in turn promotes inflammatory signaling and leukocyte migration to sites of infection. Here we investigate patterns of gene loss, diversification, and ligand recognition among FPRs in primates and carnivores. We find that FPR1, which plays a critical role in innate immune defense in humans, has been lost in New World primates. Amino acid variation in FPR1 and FPR2 among primates and carnivores is consistent with a history of repeated positive selection acting on extracellular domains involved in ligand recognition. To assess the consequences of FPR divergence on bacterial ligand interactions, we measured binding between primate FPRs and the FPR agonist Staphylococcus aureus enterotoxin B, as well as S. aureus FLIPr-like, an FPR inhibitor. We found that few rapidly evolving sites in primate FPRs are sufficient to modulate recognition of bacterial proteins, demonstrating how natural selection may serve to tune FPR activation in response to diverse microbial ligands.

Evolution of host-microbe cell adherence by receptor domain shuffling.

Stable adherence to epithelial surfaces is required for colonization by diverse host-associated microbes. Successful attachment of pathogenic microbes to host cells via adhesin molecules is also the first step in many devastating infections. Despite the primacy of epithelial adherence in establishing host-microbe associations, the evolutionary processes that shape this crucial interface remain enigmatic. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) encompass a multifunctional family of vertebrate cell surface proteins which are recurrent targets of bacterial adhesins at epithelial barriers. Here, we show that multiple members of the primate CEACAM family exhibit evidence of repeated natural selection at protein surfaces targeted by bacteria, consistent with pathogen-driven evolution. Divergence of CEACAM proteins between even closely related great apes is sufficient to control molecular interactions with a range of bacterial adhesins. Phylogenetic analyses further reveal that repeated gene conversion of CEACAM extracellular domains during primate divergence plays a key role in limiting bacterial adhesin host tropism. Moreover, we demonstrate that gene conversion has continued to shape CEACAM diversity within human populations, with abundant human CEACAM1 variants mediating evasion of adhesins from pathogenic Neisseria. Together this work reveals a mechanism by which gene conversion shapes first contact between microbes and animal hosts.

Trillions of bacteria live in and on the human body. Most of them are harmless but some can cause serious infections. To grow in or on the body, bacteria often attach to proteins on the surface of cells that make up the lining of tissues like the gut or the throat. In some cases, bacteria use these proteins to invade the cells causing an infection. Genetic mutations in the genes encoding these proteins that protect against infection are more likely to be passed on to future generations. This may lead to rapid spread of these beneficial genes in a population. A family of proteins called CEACAMs are frequent targets of infection-causing bacteria. These proteins have been shown to play a role in cancer progression. But they also play many helpful roles in the body, including helping transmit messages between cells, aiding cell growth, and helping the immune system recognize pathogens. Scientists are not sure if these multi-tasking CEACAM proteins can evolve to evade bacteria without affecting their other roles. Baker et al. show that CEACAM proteins targeted by bacteria have undergone rapid evolution in primates. In the experiments, human genes encoding CEACAMs were compared with equivalent genes from 19 different primates. Baker et al. found the changes in human and primate CEACAMs often occur through a process called gene conversion. Gene conversion occurs when DNA sections are copied and pasted from one gene to another. Using laboratory experiments, they showed that some of these changes enabled CEACAM proteins to prevent certain harmful bacteria from binding. The experiments suggest that some versions of CEACAM genes may protect humans or other primates against bacterial infections. Studies in natural populations are needed to test if this is the case. Learning more about how CEACAM proteins evolve and what they do may help scientists better understand the role they play in cancer and help improve cancer care. Studying CEACAM evolution may also help scientists understand how bacteria and other pathogens drive protein evolution in the body.

A Rapidly Evolving Polybasic Motif Modulates Bacterial Detection by Guanylate Binding Proteins.

Cell-autonomous immunity relies on the rapid detection of invasive pathogens by host proteins. Guanylate binding proteins (GBPs) have emerged as key mediators of vertebrate immune defense through their ability to recognize a diverse array of intracellular pathogens and pathogen-containing cellular compartments. Human and mouse GBPs have been shown to target distinct groups of microbes, although the molecular determinants of pathogen specificity remain unclear. We show that rapid diversification of a C-terminal polybasic motif (PBM) in primate GBPs controls recognition of the model cytosolic bacterial pathogen Shigella flexneri By swapping this membrane-binding motif between primate GBP orthologs, we found that the ability to target S. flexneri has been enhanced and lost in specific lineages of New World primates. Single substitutions in rapidly evolving sites of the GBP1 PBM are sufficient to abolish or restore bacterial detection abilities, illustrating a role for epistasis in the evolution of pathogen recognition. We further demonstrate that the squirrel monkey GBP2 C-terminal domain recently gained the ability to target S. flexneri through a stepwise process of convergent evolution. These findings reveal a mechanism by which accelerated evolution of a PBM shifts GBP target specificity and aid in resolving the molecular basis of GBP function in cell-autonomous immune defense.IMPORTANCE Many infectious diseases are caused by microbes that enter and survive within host cells. Guanylate binding proteins (GBPs) are a group of immune proteins which recognize and inhibit a variety of intracellular pathogenic microbes. We discovered that a short sequence within GBPs required for the detection of bacteria, the polybasic motif (PBM), has been rapidly evolving between primate species. By swapping PBMs between primate GBP1 genes, we were able to show that specific sequences can both reduce and improve the ability of GBP1 to target intracellular bacteria. We also show that the ability to envelop bacteria has independently evolved in GBP2 of South American monkeys. Taking the results together, this report illustrates how primate GBPs have adapted to defend against infectious pathogens.