Staphylococcus aureus adapts to the host nutritional landscape to overcome tissue-specific branched-chain fatty acid requirement

During infection, pathogenic microbes adapt to the nutritional milieu of the host through metabolic reprogramming and nutrient scavenging. For the bacterial pathogen Staphylococcus aureus, virulence in diverse infection sites is driven by the ability to scavenge myriad host nutrients, including lipoic acid, a cofactor required for the function of several critical metabolic enzyme complexes. S. aureus shuttles lipoic acid between these enzyme complexes via the amidotransferase, LipL. Here, we find that acquisition of lipoic acid, or its attachment via LipL to enzyme complexes required for the generation of acetyl-CoA and branched-chain fatty acids, is essential for bacteremia, yet dispensable for skin infection in mice. A lipL mutant is auxotrophic for carboxylic acid precursors required for synthesis of branched-chain fatty acids, an essential component of staphylococcal membrane lipids and the agent of membrane fluidity. However, the skin is devoid of branched-chain fatty acids. We showed that S. aureus instead scavenges host-derived unsaturated fatty acids from the skin using the secreted lipase, Geh, and the unsaturated fatty acid–binding protein, FakB2. Moreover, murine infections demonstrated the relevance of host lipid assimilation to staphylococcal survival. Altogether, these studies provide insight into an adaptive trait that bypasses de novo lipid synthesis to facilitate S. aureus persistence during superficial infection. The findings also reinforce the inherent challenges associated with targeting bacterial lipogenesis as an antibacterial strategy and support simultaneous inhibition of host fatty acid salvage during treatment.

The gram-positive bacterium Staphylococcus aureus is notorious for its capacity to cause widespread pathology in nearly every organ and tissue during infection (1). Much of this success can be attributed to the ability of S. aureus to extract essential nutrients from the host milieu (2). Hence, understanding the adaptive traits that allow S. aureus to exploit in situ resources for survival is critical to devising effectual treatments for staphylococcal diseases.Lipoic acid is an organosulfur compound derived from an early-stage intermediate of fatty acid biosynthesis that is required for carbon shuttling in central metabolism via a redox-sensitive dithiolane ring at its distal end (3). In a previous study, we found that shuttling of lipoic acid to metabolic enzyme complexes by the amidotransferase, LipL, was paramount for S. aureus survival in a murine model of bacteremia but not skin infection (4). At minimum, LipL appears to be required for the transfer of lipoic acid to E2 subunits of the pyruvate dehydrogenase (PDH) and branched-chain α-ketoacid dehydrogenase (BCODH) complexes (4, 5). The absence of lipoic acid renders each complex nonfunctional (4, 5). The discrepancy in demand for lipoic acid attachment in these two infection sites suggests that the nutritional environment of the skin eases the requirement for lipoic acid–dependent metabolic processes, especially those that require PDH or BCODH complex activity. PDH is responsible for the generation of acetyl-CoA upon exit from glycolysis, while BCODH is essential for the synthesis of saturated branched-chain fatty acids (BCFAs), a major component of staphylococcal membrane phospholipids (6). Like unsaturated fatty acids (UFAs), BCFAs provide membrane fluidity to staphylococci, which neither synthesize UFAs nor encode a fatty acid (FA) desaturase that converts saturated fatty acids (SFAs) to unsaturated products (7). Since a lack of membrane fluidity triggers cell death as a result of lipid phase separation and protein segregation, bacterial survival hinges on the fine-tuning of membrane lipid composition to adapt to fluctuating environments (8).Given the importance of de novo FA synthesis to ensure bacterial membrane integrity, enzymes that are involved in this metabolic process serve as attractive targets for antibacterial drug discovery. One such drug, triclosan, binds to the enoyl-acyl carrier protein reductase, FabI, a key enzyme in the type II FA synthesis (FASII) system found in archaea and bacteria (9). FASII restriction induces FA starvation and initiates the stringent response, where high concentrations of the alarmone (p)ppGpp inhibit synthesis of the FASII substrate malonyl-CoA (10). Nevertheless, mounting evidence suggests that S. aureus can resolve triclosan interference by incorporating host-derived UFAs into its membrane (11–13). During anti-FASII–adaptive outgrowth, dissipation of (p)ppGpp levels replenishes the malonyl-CoA pool and skews distribution of the FASII substrate to favor binding with FapR, a global repressor of phospholipid synthesis genes in S. aureus (10). Ultimately, this interaction sequesters FapR to allow expression of plsX and plsC, which encode enzymes that use host UFAs to synthesize phospholipids (10, 14). UFAs are commonly found esterified as triglycerides and cholesterol esters in vertebrates (15). In humans, both compounds form the lipid core of the five major groups of lipoproteins (chylomicron, VLDL, LDL, IDL, and HDL) that transport lipids throughout the body (15). Triglycerides are also the major constituents of adipocytes and exist to a lesser extent in nonadipose cells (16, 17). It has been suggested that S. aureus secretes the glycerol ester hydrolase Geh to liberate esterified FAs from triglycerides and LDL for use as FA substrates as a nutrient acquisition strategy, although this phenomenon has not yet been tested in vivo (18, 19).Despite increasing understanding of FASII bypass by S. aureus in recent years, the overall mechanism of rescue continues to be investigated. Here, we draw upon insights derived from the study of S. aureus lipoic acid synthesis and acquisition to make connections between BCFA synthesis and bacterial survival during infection. We report that de novo BCFA synthesis by S. aureus is the primary enzymatic process that necessitates lipoic acid in vitro and during skin infection. However, the requirement for BCFA synthesis in the skin is bypassed through assimilation of UFAs from host tissue. We also provide genetic, in vivo, and chemical evidence, via treatment with the over-the-counter lipase inhibitor, orlistat, that suggests UFA acquisition depends on the secreted lipase, Geh, and the UFA binding protein, FakB2. Finally, we demonstrate that ablating both BCFA synthesis and exogenous FA uptake significantly improves staphylococcal infection outcome in mice.     

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine–serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder, affecting 15 to 20% of children (1, 2). During disease flares, patients experience painful inflamed skin lesions accompanied by intense itch. Epidermal barrier dysfunction, increased type 2 immune responses, and recurrent skin infections are features of AD (3). The most common cause of infection is Staphylococcus aureus. This bacterium colonizes the skin of the majority of AD patients (4, 5). Isolates representing several S. aureus lineages are recovered from AD skin; however, strains from the clonal complex 1 (CC1) lineage are the most frequently isolated (6–9). The burden of S. aureus on lesional and nonlesional skin correlates with severity of the disease (10, 11). S. aureus directly influences pathogenesis, and several factors produced by the bacterium increase inflammation and exacerbate AD symptoms, including staphylococcal superantigen B and delta-toxin (12–15).Despite the clear association between S. aureus colonization and AD disease severity (11), the bacterial and host factor determinants underlying colonization are poorly understood (16). Adhesion is a critical early step in the colonization process. S. aureus adheres to corneocytes in the stratum corneum of AD skin (6, 17, 18). We previously found that clumping factor B (ClfB), a cell wall-anchored protein displayed on the surface of S. aureus, can mediate adhesion to corneocytes from AD patients (6). ClfB also binds to the alpha chain of fibrinogen and to the cornified envelope proteins loricrin and cytokeratin 10 (K10) in desquamated nasal epithelial cells (19–21). To date, ClfB is the only bacterial factor known to promote adherence to corneocytes in AD. However, a ClfB-deficient mutant retained the ability to bind to corneocytes (6), suggesting that additional bacterial factors are at play.Filaggrin deficiency is common in patients with established AD and is either genetic or caused by down-regulation of gene expression by Th-2–type cytokines (22–24). Filaggrin deficiency causes epidermal barrier defects and a loss of the hygroscopic filaggrin breakdown products that normally contribute to the natural moisturizing factor (NMF) in corneocytes (25). NMF comprises a collection of humectants, including filaggrin breakdown products urocanic acid and pyrrolidone acid, along with urea, citrate, lactate acid, and sugars, and is responsible for regulating hydration in the skin (26). Low-NMF levels are associated with a loss of hydration, increased disease severity, and abnormal corneocyte morphology (27). We showed recently that S. aureus binds more strongly to low-NMF AD corneocytes than to corneocytes with normal levels of NMF (18).Corneocytes with low NMF have very different surface topography when compared with corneocytes with normal levels of NMF (27). Aberrant “villus-like” projections (VPs) protrude from the surface of low-NMF corneocytes (18, 27). The protein corneodesmosin (CDSN) is confined to the cell–cell junctions between corneocytes in healthy skin, where homophilic interactions between the CDSN proteins on adjacent cells facilitate cell–cell cohesion (28). In AD patients, however, CDSN decorates the tips of the VPs on low-NMF corneocytes (27).This study aimed to elucidate a key component of S. aureus colonization by identifying the molecular determinants of adherence to AD corneocytes. We recognized that the occurrence of VPs on low-NMF corneocytes presents a different colonization surface to the bacterium and postulated that the accessibility of CDSN on the tips of VPs could influence pathogen adherence. We show that S. aureus can interact with CDSN and identify the S. aureus proteins promoting adherence to this host protein. We use single-cell and single-molecule atomic force microscopy (AFM), surface plasmon resonance (SPR), and ex vivo bacterial adherence studies with patient corneocytes to characterize this interaction. This study expands the repertoire of ligands for S. aureus and, crucially, links bacterial interactions with a host protein (CDSN) to binding to corneocytes taken from patients. Thus, our findings provide insights into the adhesion process and develop our understanding of the mechanisms underlying colonization of the skin of AD patients by S. aureus.     

Host fibrinogen drives antimicrobial function in Staphylococcus aureus peritonitis through bacterial-mediated prothrombin activation

The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib–) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib– mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.

In addition to serving as a centerpiece of hemostasis and thrombosis, fibrin(ogen) directs local inflammation in areas of tissue damage. Fibrin(ogen) can engage an array of integrin and nonintegrin cell-surface receptors to mediate downstream effector functions of innate immune cells. Whereas fibrin(ogen)-driven inflammation is detrimental in the context of inflammatory diseases like arthritis, colitis, and musculoskeletal disease (1–4), it is an important component of host defense in the context of infection (5–8). To help counter host defense mechanisms, bacteria have evolved virulence factors that engage host hemostatic system components to manipulate the activity of coagulation and fibrinolytic factors. This is particularly true for staphylococcal species, including the common, highly virulent human pathogen Staphylococcus aureus.S. aureus is a Gram-positive pathogen that expresses numerous virulence factors that engage the hemostatic system within vertebrate hosts, including an array of products that directly bind fibrin(ogen) and control fibrin deposition (9–13). S. aureus produces two nonproteolytic prothrombin activators, collectively termed “coagulases,” that bind host prothrombin and mediate fibrin formation (14, 15). Pathogen-mediated fibrin(ogen) binding and fibrin formation have been linked to S. aureus functioning as a causative agent underlying a spectrum of diseases ranging from skin infections to life-threatening pneumonia, bacteremia/sepsis, and endocarditis. A possible exception may be peritonitis. Peritoneal infection is particularly problematic for cirrhotic patients, and in hospital-acquired infections is associated with sutures, catheters, and medical implants. Intriguingly, it is coagulase-negative staphylococci (CoNS) that account for the overwhelming majority of these infections (16–20). The basis for the high prevalence of CoNS rather than the more pathogenic coagulase-positive S. aureus, and whether there is a functional link to bacterial-driven prothrombin activation, remains undefined. Here, we explored the hypothesis that host fibrinogen and prothrombin drive a bacterial killing mechanism and defined the contribution of bacterial coagulases to infection in a murine model of acute S. aureus peritonitis.     

Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYO^Sa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYO^Sa. Because of the mode of action of PYO^Sa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYO^Sa; none were observed in the 13 clinical isolates tested. PYO^Sa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYO^Sa and antibiotics raise issues that must be addressed before PYO^Sa is employed clinically. Despite the maintenance of the phage, PYO^Sa does not clear populations of S. aureus. Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise–resurrection dynamics of PYO^Sa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYO^Sa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.

Driven by well-warranted concerns about the increasing frequencies of infections with antibiotic-resistant pathogens, there has been a resurrection of interest in, research on, and clinical trials with a therapy that predates antibiotics by more than 15 y: bacteriophage (1–4). One direction phage therapy research has taken is to engineer lytic (virulent) phages with properties that are anticipated to maximize their efficacy for treating bacterial infections in mammals (5–8). Primary among these properties are 1) a broad host range for the target bacterial species; 2) mechanisms that prevent the generation of envelope or other kinds of high-fitness resistance in the target bacteria (9); 3) the capacity to thwart the innate and adaptive immune systems of bacteria, respectively, restriction-modification and CRISPR-Cas (7, 10, 11); 4) the ability to survive, kill, and replicate on pathogenic bacteria colonizing or infecting mammalian hosts (12, 13); and 5) little or no negative effects on the treated host (9).To these five desired properties for therapeutic bacteriophages, there is a sixth that should be considered: the joint action of these phages and antibiotics (8, 14–17). Phages-only treatment may be reasonable for compassionate therapy, where the bacteria responsible for the infection are resistant to all available antibiotics (18–20). From a practical perspective, however, for phages to become widely employed for treating bacterial infections, they would have to be effective in combination with antibiotics. It would be unethical and unacceptable to clinicians and regulatory agencies to use phage-only therapy for infections that can be effectively treated with existing antibiotics.Although not specifically engineered for these properties, there is a Staphylococcal phage isolated from a therapeutic phage collection from the Eliava Institute in Tbilisi, Georgia, that we call PYO^Sa that on first consideration appears to have all six of the properties required to be an effective agent for therapy. 1) PYO^Sa is likely to have a broad host range for S. aureus. The receptor of this K-like Myoviridae is N-acetylglucosamine in the wall-teichoic acid backbone of Staphylococcus aureus and is shared among most (21), if not all, S. aureus, thereby suggesting PYO^Sa should be able to adsorb to and potentially replicate on and kill a vast number of clinical isolates of S. aureus. 2) S. aureus does not generate classical, surface modification mutants resistant to PYO^Sa. Since the structure of the receptor of PYO^Sa is critical to the viability, replication, and virulence of these bacteria, the modifications in this receptor (22) may not be consistent with the viability or pathogenicity of S. aureus (23). 3) The replication of PYO^Sa is unlikely to be prevented by restriction-modification (RM) or CRISPR-Cas. Despite a genome size of 127 KB, the PYO^Sa phage has no GATC nucleotide restriction sites for the S. aureus restriction enzyme Sau3A1 and only one restriction site, GGNCC (guanine, guanine, any nucleotide, cytosine, cytosine), for the Sau961 restriction endonuclease (24, 25). There is no evidence for a functional CRISPR-Cas system in S. aureus or, to our knowledge, other mechanisms by which S. aureus may prevent the replication of this phage (26). 4) There is evidence that PYO^Sa-like phages can replicate in mammals. Early treatment with a phage with a different name but the same properties as PYO^Sa, Statu^v, prevented mortality in otherwise lethal peritoneal infections of S. aureus in mice (27). A PYO^Sa-like phage has also been successfully used therapeutically in humans (28). 5) No deleterious effects of a PYO^Sa-like phage were observed in recent placebo-controlled trials with volunteers asymptotically colonized by S. aureus (24). 6) Finally, there is evidence to suggest synergy with antibiotics. In vitro, PYO^Sa increased the efficacy of low concentrations of antibiotics for the treatment of biofilm populations of S. aureus (14).With in vitro population and evolutionary dynamic experiments with PYO^Sa and S. aureus Newman in combination with three different bacteriostatic and six different bactericidal antibiotics, we explore just how well PYO^Sa fits the above criteria for combination antibiotic and phage therapy. Our population dynamic experiments indicate that as consequence of the ascent of potentially pathogenic PYO^Sa resistant small colony variants (SCVs), by itself, PYO^Sa does not clear S. aureus infections. After an initial decline in the density of these bacteria when confronted with PYO^Sa, despite the continued presence of these phage, the densities of the bacterial populations return to levels similar to those observed in their absence. Using mathematical models, we present a hypothesis to account for these demise resurrection population dynamics, and the continued maintenance of the phage following the ascent of PYO^Sa resistant SCVs. We test and provide evidence in support of that hypothesis with a DNA sequence analysis of the genetic basis of the SCVs. By combining PYO^Sa with antibiotics, the density of the S. aureus population can be markedly reduced. There are, however, significant differences in the effectiveness of this combination therapy depending on whether the antibiotics and phage are used simultaneously or sequentially and on whether the antibiotics are bacteriostatic or bactericidal. Treatment with PYO^Sa, followed by the administration of bactericidal antibiotics, is more effective in reducing density of these bacterial population than treatment with these antibiotics alone. The methods developed here to evaluate the clinical potential of PYO^Sa in combination with antibiotics and design protocols for treating S. aureus infections with these phages and antibiotics can be employed for these purposes for any phage and bacterium that can be cultured in vitro.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Structure,self-assembly,and properties of a truncated reflectin variant

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between the protein’s structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Materials from naturally occurring and recombinant proteins are frequently employed for the study of fundamental biological processes and leveraged for applications in fields as diverse as electronics, optics, bioengineering, medicine, and fashion (1–13). Such broad utility is enabled by the numerous advantageous characteristics of protein-based materials, which include sequence modularity, controllable self-assembly, stimuli-responsiveness, straightforward processability, inherent biological compatibility, and customizable functionality (1–13). Within this context, unique structural proteins known as reflectins have recently attracted substantial attention because of their key roles in the fascinating color-changing capabilities of cephalopods, such as the squid shown in Fig. 1A, and have furthermore demonstrated their utility for unconventional biophotonic and bioelectronic technologies (11–40). For example, in vivo, Bragg stack-like ultrastructures from reflectin-based high refractive index lamellae (membrane-enclosed platelets) are responsible for the angle-dependent narrowband reflectance (iridescence) of squid iridophores, as shown in Fig. 1B (15–20). Analogously, folded membranes containing distributed reflectin-based particle arrangements within sheath cells lead to the mechanically actuated iridescence of squid chromatophore organs, as shown in Fig. 1C (15, 16, 21, 22). Moreover, in vitro, films processed from squid reflectins not only exhibit proton conductivities on par with some state-of-the-art artificial materials (23–27) but also support the growth of murine and human neural stem cells (28, 29). Additionally, morphologically variable coatings assembled from different reflectin isoforms can enable the functionality of chemically and electrically actuated color-changing devices, dynamic near-infrared camouflage platforms, and stimuli-responsive photonic architectures (27, 30–34). When considered together, these discoveries and demonstrations constitute compelling motivation for the continued exploration of reflectins as model biomaterials.Open in a separate windowFig. 1.(A) A camera image of a D. pealeii squid for which the skin contains light-reflecting cells called iridophores (bright spots) and pigmented organs called chromatophores (colored spots). Image credit: Roger T. Hanlon (photographer). (B) An illustration of an iridophore (Left), which shows internal Bragg stack-like ultrastructures from reflectin-based lamellae (i.e., membrane-enclosed platelets) (Inset). (C) An illustration of a chromatophore organ (Left), which shows arrangements of reflectin-based particles within the sheath cells (Inset). (D) The logo of the 28-residue-long N-terminal motif (RMN), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (E) The logo of the 28-residue-long internal motif (RMI), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (F) The logo of the 21-residue-long C-terminal motif (RMC), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (G) The amino acid sequence of full-length D. pealeii reflectin A1, which contains a single RMN motif (gray oval) and five RMI motifs (orange ovals). (H) An illustration of the selection of the prototypical truncated reflectin variant (denoted as RfA1TV) from full-length D. pealeii reflectin A1.Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures (30, 31, 35–39). For example, fibers drawn from full-length Euprymna scolopes reflectin 1a and films processed from truncated E. scolopes reflectin 1a were shown to possess secondary structural elements (i.e., α-helices or β-sheets) (30, 31). In addition, precipitated nanoparticles and drop-cast films from full-length Doryteuthis pealeii reflectin A1 have exhibited β-character, which was seemingly associated with their conserved motifs (35, 36). Moreover, nanoparticles assembled from both full-length and truncated Sepia officinalis reflectin 2 variants have demonstrated signatures consistent with β-sheet or α-helical secondary structure, albeit in the presence of surfactants (38). However, such studies were made exceedingly challenging by reflectins’ atypical primary sequences enriched in aromatic and charged residues, documented extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for poorly controlled aggregation (12, 14, 15, 30–32, 34–39). Consequently, the reported efforts have all suffered from multiple drawbacks, including the need for organic solvents or denaturants, the evaluation of only polydisperse or aggregated (rather than monomeric) proteins, a lack of consensus among different experimental techniques, inadequate resolution that precluded molecular-level insight, imperfect agreement between computational predictions and experimental observations, and/or the absence of conclusive correlations between structure and optical functionality. As such, there has emerged an exciting opportunity for investigating reflectins’ molecular structures, which remain poorly understood and the subject of some debate.Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a robust methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between its structural characteristics and optical properties. We first rationally select a prototypical reflectin variant expected to recapitulate the behavior of its parent protein by using a bioinformatics-guided approach. We next map the conformational and energetic landscape accessible to our selected protein by means of all-atom molecular dynamics (MD) simulations. We in turn produce our truncated reflectin variant with and without isotopic labeling, develop solution conditions that maintain the protein in a monomeric state, and characterize the variant’s size and shape with small-angle X-ray scattering (SAXS). We subsequently resolve our protein’s dynamic secondary and tertiary structures and evaluate its backbone conformational fluctuations with NMR spectroscopy. Finally, we demonstrate a straightforward mechanical agitation-based approach to controlling our truncated reflectin variant’s secondary structure, hierarchical self-assembly, and bulk refractive index distribution. Overall, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and appear poised to inform new directions across biochemistry, cellular biology, bioengineering, and optics.     

The feeding system of Tiktaalik roseae: an intermediate between suction feeding and biting

Changes to feeding structures are a fundamental component of the vertebrate transition from water to land. Classically, this event has been characterized as a shift from an aquatic, suction-based mode of prey capture involving cranial kinesis to a biting-based feeding system utilizing a rigid skull capable of capturing prey on land. Here we show that a key intermediate, Tiktaalik roseae, was capable of cranial kinesis despite significant restructuring of the skull to facilitate biting and snapping. Lateral sliding joints between the cheek and dermal skull roof, as well as independent mobility between the hyomandibula and palatoquadrate, enable the suspensorium of T. roseae to expand laterally in a manner similar to modern alligator gars and polypterids. This movement can expand the spiracular and opercular cavities during feeding and respiration, which would direct fluid through the feeding apparatus. Detailed analysis of the sutural morphology of T. roseae suggests that the ability to laterally expand the cheek and palate was maintained during the fish-to-tetrapod transition, implying that limited cranial kinesis was plesiomorphic to the earliest limbed vertebrates. Furthermore, recent kinematic studies of feeding in gars demonstrate that prey capture with lateral snapping can synergistically combine both biting and suction, rather than trading off one for the other. A “gar-like” stage in early tetrapod evolution might have been an important intermediate step in the evolution of terrestrial feeding systems by maintaining suction-generation capabilities while simultaneously elaborating a mechanism for biting-based prey capture.

Although suction feeding is a primary mode of prey capture among aquatic vertebrates (1), it is physically impractical on land due to the lower viscosity of air as compared to water (2–4). Terrestrial-feeding vertebrates must resort to other means, such as biting or tongue capture, to procure food (2). Naturally, researchers seeking to understand shifts in feeding strategies in tetrapodomorph vertebrates during the water-to-land transition have focused primarily on whether feeding systems in fossil forms showed adaptations for either suction or biting (2, 5). Generally, plesiomorphic “fish-like” morphology is interpreted as a means to create suction during the feeding cycle, and derived “tetrapod-like” morphology is interpreted as suggestive of biting (5–8). Suction feeding in fish is typically associated with jointed, kinetic skulls that allow for large volumetric expansion to draw in food (1, 9). In contrast, many lineages of modern tetrapods have consolidated skulls, such as mammals, crocodilians, and amphibians, thought to strengthen the skull for biting (9–12). While there is evidence for kinetic joints in the palate and skull roof of multiple early stem tetrapods (6, 13–16), it is uncertain if they represent plesiomorphic holdovers of limited fish-like cranial kinesis (13, 17, 18) or were independently derived mechanisms to improve biting capabilities on land (17, 19).A central challenge of paleontology has been to understand how, and when, transitions in the feeding system of early terrestrial vertebrates occurred. Late Devonian finned tetrapodomorphs, typified by Eusthenopteron foordi, have expansive, kinetic skulls with open sutures, robust gill covers, large hyomandibulae, tall palatal elements, and a jointed neurocranium all thought to be features that play a role in suction feeding (5, 9, 20). In contrast, the Late Devonian limbed tetrapodomorph Acanthostega gunnari has a flat skull, interdigitating sutures between the bones of the skull roof, absent gill covers, reduced hyomandibulae, horizontal palatal elements, and a consolidated neurocranium that are hypothesized to be derived adaptations for biting (5, 6, 21, 22). Analyses of tetrapodomorph lower jaws have produced equivocal results, noting few differences between presumed aquatic and terrestrial forms (7, 8). These results suggest that either a fish-like suction-based feeding mechanism was maintained well into the Carboniferous (7, 8, 23) or that a biting-based feeding mechanism had evolved in water prior to the origin of terrestrial tetrapods (24).To understand how feeding modes shifted among tetrapodomorphs and assess the origin of novel feeding mechanisms in the tetrapod lineage, we use high-resolution microcomputed tomography (µCT) to analyze multiple specimens of a well-preserved elpistostegalian-grade tetrapodomorph, Tiktaalik roseae, and compare the anatomy resolved from those µCT scans to features of other extinct tetrapodomorphs and extant fishes with analogous features. T. roseae is a tetrapodomorph from the Upper Devonian (Frasnian, ∼375 Mya) of Arctic Canada (Ellesmere Island, Nunavut Territory) (25, 26) that, according to most-recent phylogenies (27, 28), is representative of the outgroup of limbed vertebrates (tetrapods). Although plesiomorphic in lower jaw morphology (7, 8, 29), elpistostegalian-grade tetrapodomorphs (a group also including Panderichthys rhombolepis and Elpistostege watsoni) represent a period of rapid cranial evolution that could nevertheless suggest shifts in feeding strategies (5, 26, 30, 31). µCT was performed on four specimens of T. roseae from the Nunavut Fossil Vertebrate Collection (NUFV) consisting of three-dimensionally (3D) preserved palatal material in articulation with the cranium, as well as individual bones from multiple disarticulated specimens (SI Appendix, Table S1). Sutural cross-sections were compared with homologous sutures reported for E. foordi (5, 20) and A. gunnari (5, 21). Cranial joints were compared with possible modern analogs, alligator gar (Atractosteus spatula) (32–34) and ornate bichir (Polypterus ornatipinnis) (5, 32, 35), which were selected on the basis of convergent feeding morphologies with T. roseae. Finally, joints between the palate, hyomandibula, and braincase were modeled with the same kinematic range of motion as reported in A. spatula for comparison purposes (34).