Hydrogen sulfide (H2S) is a respiratory toxicant that creates extreme environments tolerated by few organisms. H2S is also produced endogenously by metazoans and plays a role in cell signaling. The mechanisms of H2S toxicity and its physiological functions serve as a basis to discuss the multifarious strategies that allow animals to survive in H2S-rich environments. Despite their toxicity, H2S-rich environments also provide ecological opportunities, and complex selective regimes of covarying abiotic and biotic factors drive trait evolution in organisms inhabiting H2S-rich environments. Furthermore, adaptation to H2S-rich environments can drive speciation, giving rise to biodiversity hot spots with high levels of endemism in deep-sea hydrothermal vents, cold seeps, and freshwater sulfide springs. The diversity of H2S-rich environments and their inhabitants provides ideal systems for comparative studies of the effects of a clear-cut source of selection across vast geographic and phylogenetic scales, ultimately informing our understanding of how environmental stressors affect ecological and evolutionary processes.

Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)—a toxicant that impairs mitochondrial function—across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and—in some instances—codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

Chlamydia trachomatis is an obligate intracellular bacterium that infects the oculogenital mucosae. C. trachomatis infection of the eye causes trachoma, the leading cause of preventable blindness. Infections of the genital mucosae are a leading cause of sexually transmitted diseases. A vaccine to prevent chlamydial infection is needed but has proven difficult to produce by using conventional vaccination approaches. Potent immunity to vaginal rechallenge in a murine model of chlamydial genital infection has been achieved only by infection or by immunization with dendritic cells (DC) pulsed ex vivo with whole inactivated organisms. Immunity generated by infection or ex vivo antigen-pulsed DC correlates with a chlamydia-specific interleukin 12 (IL-12)-dependent CD4 ؉ Th1 immune response. Because of the potent antichlamydial immunizing properties of DC, we hypothesized that DC could be a powerful vehicle for the delivery of individual chlamydial antigens that are thought to be targets for more conventional vaccine approaches. Here, we investigated the recombinant chlamydial major outer membrane protein (rMOMP) as a target antigen. The results demonstrate that DC pulsed with rMOMP secrete IL-12 and stimulate infection-sensitized CD4 ؉ T cells to proliferate and secrete gamma interferon. These immunological properties implied that rMOMP-pulsed DC would be potent inducers of MOMP-specific CD4 ؉ Th1 immunity in vivo; however, we observed the opposite result. DC pulsed ex vivo with rMOMP and adoptively transferred to naive mice generated a Th2 rather than a Th1 anti-MOMP immune response, and immunized mice were not protected following infectious challenge. We conclude from these studies that the immunological properties of ex vivo pulsed DC are not necessarily predictive of the immune response generated in vivo following adoptive transfer. These findings suggest that the nature of the antigen used to pulse DC ex vivo influences the Th1-Th2 balance of the immune response in vivo.

The notorious plasticity of gene expression responses and the complexity of environmental gradients complicate the identification of adaptive differences in gene regulation among populations. We combined transcriptome analyses in nature with common-garden and exposure experiments to establish cause-effect relationships between the presence of a physiochemical stressor and expression differences, as well as to test how evolutionary change and plasticity interact to shape gene expression variation in natural systems. We studied two evolutionarily independent population pairs of an extremophile fish (Poecilia mexicana) living in toxic, hydrogen sulphide (H S)-rich springs and adjacent nontoxic habitats and assessed genomewide expression patterns of wild-caught and common-garden-raised individuals exposed to different concentrations of H S. We found that 7.7% of genes that were differentially expressed between sulphidic and nonsulphidic ecotypes remained differentially expressed in the laboratory, indicating that sources of selection other than H S-or plastic responses to other environmental factors-contribute substantially to gene expression patterns observed in the wild. Concordantly differentially expressed genes in the wild and the laboratory were primarily associated with H S detoxification, sulphur processing and metabolic physiology. While shared, ancestral plasticity played a minor role in shaping gene expression variation observed in nature, we documented evidence for evolved population differences in the constitutive expression as well as the H S inducibility of candidate genes. Mechanisms underlying gene expression variation also varied substantially across the two ecotype pairs. These results provide a springboard for studying evolutionary modifications of gene regulatory mechanisms that underlie expression variation in locally adapted populations.

Chlamydia trachomatis is an obligate intracellular pathogen with global health and economic impact. Upon infection, C. trachomatis resides within a protective niche, the inclusion, wherein it replicates and usurps host cell machinery and resources. The inclusion membrane is the key host-pathogen interface that governs specific protein-protein interactions to manipulate host signaling pathways. At the conclusion of the infection cycle, C. trachomatis exits the host cell via lysis or extrusion. Extrusion depends on the phosphorylation state of myosin light chain 2 (MLC2); the extent of phosphorylation is determined by the ongoing opposing activities of myosin phosphatase (MYPT1) and myosin kinase (MLCK). Previously, it was shown that MYPT1 is recruited to the inclusion and interacts with CT228 for regulation of host cell egress. In this study, we generated a targeted chromosomal mutation of CT228 (L2-ΔCT228) using the TargeTron system and demonstrate a loss of MYPT1 recruitment and increase in extrusion production in vitro. Mutation of CT228 did not affect chlamydial growth in cell culture or recruitment of MLC2. Moreover, we document a delay in clearance of L2-ΔCT228 during murine intravaginal infection as well as a reduction in systemic humoral response, relative to L2-wild type. Taken together, the data suggest that loss of MYPT1 recruitment (as a result of CT228 disruption) regulates the degree of host cell exit via extrusion and affects the longevity of infection in vivo.

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We studied the expression of cytokines, chemokines, and chemokine receptors by the RNase protection assay in chlamydia-pulsed dendritic cells to better understand their potent anti-chlamydial immunizing properties. We found that chlamydia-pulsed dendritic cells express a complex profile of inflammatory and immunomodulatory molecules. These include CCR-7, interleukin-12, and interferon-induced protein 10, molecules that might influence the homing of pulsed dendritic cells to the site of chlamydial infection and the induction of a local protective CD4 ؉ Th1 cellular immunity.Chlamydia trachomatis is an obligatory intracellular bacterial parasite that infects the oculogenital mucosal epithelium, causing trachoma, the world's leading cause of preventable blindness, and sexually transmitted diseases. Pelvic inflammatory disease is a serious sequalae of C. trachomatis infection of the female genital tract that can result in tubal blockage, infertility, or ectopic pregnancy (2,4,8,13). The development of an efficacious vaccine against C. trachomatis oculogenital infection is likely to be key to the control of both trachoma and chlamydial sexually transmitted diseases. Despite considerable effort, however, there has been little favorable progress toward this end. Conventional vaccination approaches have produced disappointing results in their abilities to prevent infection of the mouse female genital tract (12,14,19), despite a modicum of success in controlling chlamydial infection of the respiratory tract (20). Solid protective immunity to genital rechallenge has been achieved only by infection or adoptive immunization with dendritic cells (DC) pulsed ex vivo with inactivated whole chlamydial organisms (6, 18). Interestingly, mice immunized with chlamydia-pulsed DC exhibit equivalent levels of protective immunity to that in mice that have spontaneously resolved a primary genital infection (18). Both infection-mediated protective immunity and immunity elicited following adoptive transfer of antigen-pulsed DC correlate with a chlamydia-specific CD4ϩ Th1-biased immune response characterized by the secretion of high levels of gamma interferon from local and splenic CD4ϩ T cells (6,18). Recent studies have also indicated an important cooperative role for both CD4 ϩ T cells and B cells in recall immunity in the murine model; however the mechanism(s) that mediates this cooperative effector function has not been described (11). Clearly, the use of ex vivo antigenpulsed DC as a practical chlamydial vaccine is unsuited for use in humans. Nevertheless, the ability of ex vivo antigen-pulsed DC to elicit solid antichlamydial protective immunity at the genital mucosae is gratifying because it demonstrates that a more complete understanding of chlamydia-DC interactions may provide important information applicable to the development of a conventional antichlamydial vaccine.In this work we have investigated cytokine, chemokine, and chemokine receptor gene expression in chlamydia-pulsed DC by the RNase protection assay (RPA). Our findin...

22Extreme environments test the limits of life. Still, some organisms thrive in harsh conditions, 23 begging the question whether the repeated colonization of extreme environments is facilitated by 24 predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying 25 convergent evolution of tolerance to hydrogen sulfide (H2S)-a potent toxicant that impairs 26 mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, 27 Poeciliidae) from H2S-rich freshwater springs. We found that mitochondrial function is maintained 28 in the presence of H2S in sulfide spring P. mexicana, but not ancestral lineages in adjacent nonsulfidic 29 habitats, due to convergent adaptations in both the primary toxicity target and a major detoxification 30 enzyme. Additionally, we show that H2S tolerance in 10 independent lineages of sulfide spring fishes 31 across multiple genera of Poeciliidae is mediated by convergent modification and expression changes 32 of genes associated with H2S toxicity and detoxification. Our results demonstrate that the repeated 33 modification of highly conserved physiological pathways associated with essential mitochondrial 34 processes enabled the colonization of novel environments. 35 36 3 Stephen J. Gould was a fierce proponent of the importance of contingency in evolution, famously 37 quipping that replaying the "tape of life" would lead to different outcomes every time (1). 38Mitochondrial genomes were historically thought to be a prime example of such contingency 39 evolution, because alternative genetic variants were assumed to be selectively neutral (2). This 40 paradigm has been shifting, with mounting evidence that mitochondria-and genes encoded in the 41 mitochondrial genome-play an important role in adaptation, especially in the context of 42 physiochemical stress (3). However, it often remains unclear how genetic variation in mitochondrial 43 genomes and nuclear genes that contribute to mitochondrial function translates to variation in 44 physiological and organismal function. Furthermore, it is not known whether exposure to similar 45 selective regimes may cause convergent modifications of mitochondrial genomes and emergent 46 biochemical and physiological functions in evolutionarily independent lineages. Extreme 47 environments that represent novel ecological niches are natural experiments to address questions 48 about mechanisms underlying mitochondrial adaptations and illuminate the predictability of adaptive 49 evolution of mitochondria. Among the most extreme freshwater ecosystems are springs with high 50 levels of hydrogen sulfide (H2S), a potent respiratory toxicant lethal to metazoans due to its 51 inhibition of mitochondrial ATP production (4). Multiple lineages of livebearing fishes (Poeciliidae) 52 have colonized H2S-rich springs throughout the Americas and independently evolved tolerance to 53 sustained H2S concentrations orders of magnitudes higher than those encountered by ancestral 54 lineages in nonsulfid...