Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries)

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1β, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Heart failure is a globally significant problem in both humans and lower animals.^3,18 The medical literature is replete with predisposing causes of heart disease,¹³ yet the prevalence of heart failure remained high.^4,5,16 Regardless of the cause of myocardial damage and subsequent left ventricular compromise, the literature indicated that the proinflammatory response that occurs after myocardial infarction is an important contributor to the deterioration of the myocardium^{1,9,12,14,17,18,20,21} Sheep and pigs are excellent translational models of human cardiology because their hearts bear many physiologic and anatomic similarities to the human heart.^4,8,15 The primary use of these models in cardiology is primarily to study myocardial infarction^5,13,16 and to a lesser extent, physiologic processes that develop after myocardial insult.Our study measured some of the major proinflammatory cytokines that contribute to myocardial damage. Most of these cytokines, including: TNFα, IL6, and IFNγ, are important correlates of myocardial ischemia that contribute to a decline in left ventricular myocardial function.^1,9,14 In our study, we detected left ventricular compromise as early as 4 wk after the infarction, while the proinflammatory response was recorded at 48 h after the infarct and peaked at 4 wk. Cardiac functional parameters began to decline early in the study consistent with the proinflammatory response. The cardiac functional parameters continued to decline until 3 mo, which was the termination of the study. These findings may support antiinflammatory intervention as an important adjunct of any therapeutic regimen.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

The Halophyte Seashore Paspalum Uses Adaxial Leaf Papillae for Sodium Sequestration

Salinity is a growing issue worldwide, with nearly 30% of arable land predicted to be lost due to soil salinity in the next 30 years. Many grass crops that are vital to sustain the world’s caloric intake are salt sensitive. Studying mechanisms of salt tolerance in halophytic grasses, plants that thrive in salt conditions, may be an effective approach to ultimately improve salt-sensitive grass crops. Seashore paspalum (Paspalum vaginatum) is a halophytic Panicoid grass able to grow in salt concentrations near that of seawater. Despite its widespread cultivation as a sustainable turfgrass, the mechanism underlying its ability to retain high Na⁺ concentrations in photosynthetic tissue while maintaining growth remains unknown. We examined the leaf structure and ion content in P. vaginatum ‘HI10’, which shows increased growth under saline conditions, and Paspalum distichum ‘Spence’, which shows reduced growth under salt, to better understand the superior salt tolerance of cv HI10. A striking difference between cv HI10 and cv Spence was the high steady-state level of K⁺ in cv HI10. Imaging further showed that the adaxial surface of both cv HI10 and cv Spence contained dense costal ridges of papillae. However, these unicellular extensions of the epidermis were significantly larger in cv HI10 than in cv Spence. The cv HI10 papillae were shown to act as Na⁺ sinks when plants were grown under saline conditions. We provide evidence that leaf papillae function as specialized structures for Na⁺ sequestration in P. vaginatum, illustrating a possible path for biotechnological improvement of salt-sensitive Panicoid crops with analogous leaf structures.

About 20% of irrigated land is considered saline, with the amount of saline soils increasing worldwide (Mayak et al., 2004). This is due to increased irrigation in agricultural fields necessitated by more frequent droughts due to climate change. This trend is alarming due to the high salt sensitivity of most crop species that we rely on for vital resources. Yield reduction in crops in saline soils amounts to losses on the order of 12 to 27.3 billion U.S. dollars annually (Qadir et al., 2014). Thus, the improvement of salt tolerance in plants will become key in the coming decades. Breeding salt-tolerant crops is a cost-effective approach to improve growth in saline soils. Although much work has focused on breeding salt-tolerant species, progress in this area has been slow due to the complex genetic and physiological nature of the salt response. Furthermore, most research has been conducted on glycophytic model systems that are salt sensitive (Munns and Gilliham, 2015). Unraveling the salt-tolerance mechanisms in halophytes, species that can complete their life cycle in 200 mm salt concentrations, and transferring these pathways into glycophytes is therefore of great interest (Rajalakshmi and Parida, 2012; Roy and Chakraborty, 2014).Both glycophytes and halophytes have evolved a multitude of salt-tolerance mechanisms, including sodium (Na⁺) exclusion, sequestration, and secretion; osmolyte production; ion homeostasis; and reactive oxygen species (ROS) detoxification (Meng et al., 2018). Often, mechanisms present in glycophytes, such as osmolyte production and Na⁺ exclusion, are utilized in halophytes at higher efficiency (Wyn Jones and Storey, 1981; Grieve and Maas, 1984). However, halophytes also use mechanisms that are absent in glycophytes. Salt sequestration and secretion via salt glands is a halophyte-specific mechanism of coping with salt (Flowers and Colmer, 2008). Salt glands are found in over 50 species in 14 angiosperm families with four subtypes: epidermal bladder cells, complex multicellular glands, bicellular glands, and unicellular glands (Dassanayake and Larkin, 2017). The Poales order contains ∼8% of all halophytes (Flowers et al., 2010) and has therefore been the focus of much salt-gland-focused work (Ceccoli et al., 2015). As salt tolerance has independently evolved >70 times in grass lineages (Bennett et al., 2013), studying these salt sequestering/secreting structures in grasses is an excellent approach to better understand salt tolerance mechanisms in halophytes.Most structural and physiological work on salt glands in grasses has been conducted in the Chloridoideae and Oryzoideae subfamilies. Grasses carry either unicellular or bicellular glands, often referred to as glandular trichomes or microhairs, on the leaf surface (Dassanayake and Larkin, 2017). Microhairs have been observed on the leaf surface in all grass subfamilies except the Pooideae, and have evolved diverse functions including the sequestration or secretion of substances such as callose and heavy metals (Burke et al., 2000; Ceccoli et al., 2015). Unicellular structures on the adaxial leaf side able to secrete salt are only found in the Oryzoideae wild rice species Porteresia coarctata (Flowers et al., 1990; Sengupta and Majumder, 2009). Salt glands in the Chloridoideae are bicellular, consisting of a cap cell and a lower basal cell, both of which are dense in cytoplasm and mitochondria (Ceccoli et al., 2015). The cuticle is thickened above the cap cell in some species, forming a cuticular chamber used for storing secreted salts (Amarasinghe and Watson, 1988). In the Panicoideae, a few cases of Na⁺ secretion have been reported (McWhorter et al., 1995; Ramadan and Flowers, 2004), but to date, no sequestration structures have been identified.The Panicoideae subfamily includes the agronomically important food crops maize (Zea mays) and sorghum (Sorghum bicolor) in addition to the biofuel grasses miscanthus (Miscanthus sinensis), switchgrass (Panicum virgatum), and sugarcane (Saccharum officinarum). One of the most salt-tolerant species in the Panicoideae is the halophyte seashore paspalum (Paspalum vaginatum). It is cultivated as a turfgrass worldwide and derives its popularity from its ability to be irrigated with brackish water. P. vaginatum can survive in salt concentrations near that of seawater (Uddin et al., 2012) and uses osmolyte production, ion homeostasis, and Na⁺ exclusion to cope with salt stress (Peacock and Dudeck, 1985; Lee et al., 2008; Guo et al., 2016). However, its ability to maintain growth while accumulating high levels of Na⁺ in leaf tissue remains perplexing.Here, we studied the leaf structure and Na⁺ sequestration in ‘HI10’, a P. vaginatum cultivar, and ‘Spence’, a Paspalum distichum cultivar. P. vaginatum and P. distichum are closely related (and possibly the same species; Eudy et al., 2017), and constitute group “Disticha” in the tribe Paspaleae. P. distichum is less salt tolerant than P. vaginatum and is typically found in freshwater habitats (Leithead et al., 1971). P. vaginatum and P. distichum therefore represent a useful species pair to study salt tolerance. Furthermore, their salt responses can be compared with those of sorghum, a Panicoid glycophyte. Our main research objective was to identify the phenotypic and physiological factors that contribute to the differential tolerance to salt stress of the two Paspalum spp. cultivars and sorghum ‘BTx623’. We show that both Paspalum species contain dense rows of translucent papillae on the adaxial surface. The papillae are unicellular protrusions from epidermal cells and are much larger in cv HI10 than in cv Spence. We further demonstrate that the papillae sequester Na⁺ under salt stress. This study thus provides evidence of Na⁺ sequestration in specialized leaf-borne organs within the Panicoideae.     

S2 domain gives myosin filaments some flexibility

JGP microscopy study supports the idea that the region linking myosin head and tail domains can be peeled away from filament backbone to prevent actin-attached heads from impeding filament movement.

Myosin II motors move along actin filaments by coupling cycles of ATP binding and hydrolysis to a repetitive process in which the myosin head domains attach to actin, undergo a conformational shift/powerstroke, and then detach. In muscle cells, myosin II molecules assemble into thick filaments containing hundreds of head domains, and any heads that remain attached to actin after completing their power stroke may impede the ability of other heads to move the filament and drive muscle contraction. In this issue of JGP, Brizendine et al. provide direct evidence that this potential drag on filament movement is limited by the flexibility of myosin II’s S2 subdomain (1).(Left to right) Richard Brizendine, Christine Cremo, and Murali Anuganti provide direct evidence that the S2 domain of myosin II is a flexible structure, which would allow it to prevent actin-attached heads from impeding the movement of myosin filaments. Quantum dots labeling a head domain (black) and the filament backbone (red) mostly follow the same trajectory as a filament moves in vitro. But, in rare instances (insets), an actin-attached head briefly lags the backbone’s trajectory before catching up, an event facilitated by the flexibility of the S2 region that connects the motor protein’s head and tail domains.For the past few years, Christine Cremo and colleagues at the University of Nevada, Reno, have been studying the kinetics of filament movement using fluorescently labeled myosin and actin filaments in vitro (2). Based on their data, Cremo’s team, in collaboration with Josh Baker, developed a mixed kinetic model that predicted a key mechanical function for the S2 subdomain of myosin II, which links the motor protein’s head domains to the C-terminal light meromyosin (LMM) domains that mediate filament assembly (3,4). According to the model, the flexibility of the S2 subdomain, and its ability to be peeled away from the filament backbone, provides some slack to actin-attached heads as the filament moves forward, giving them more time to detach before they impede the filament’s progress.“So now we wanted to see if we could directly observe this flexibility,” Cremo explains. To do this, two postdocs in Cremo’s laboratory, Richard Brizendine and Murali Anuganti, assembled smooth muscle myosin filaments labeled with two differently colored quantum dots, one attached to the LMM domain and the other attached to the head domain. Most of the time, these two labels should follow the same trajectory along actin filaments in vitro. If the S2 domain is flexible, however, it should be possible to occasionally observe an actin-attached head remain in place while the LMM domain continues moving forward. This brief “dwell” should then be followed by a “jump” as the head domain detaches from actin and catches up with the trajectory of the filament backbone.“We were looking for rare events in a sea of noise,” Cremo says, yet the researchers were able to identify dwells and jumps in the quantum dot trajectories consistent with the predicted flexibility of the S2 domain. The frequency and duration of these events fit the known kinetics of actomyosin motility.Based on their data, Brizendine et al. (1) estimate that, in smooth muscle, a myosin filament can move up to ∼52 nm without being impeded by an actin-attached head, a figure close to that predicted by the mixed kinetic model. To provide this flexibility, the researchers calculate that as much as 26 nm of the S2 domain can be unzipped from the filament backbone. Intriguingly, this matches the maximum length that S2 can be seen to project from thick filaments in tomograms of Drosophila flight muscle (5), and the forces generated by working myosin heads should be more than sufficient to achieve this unzipping.Many cardiomyopathy-associated mutations are located in the S2 region of myosin II. However, the mixed kinetic model predicts that, compared with smooth muscle, myosin filaments in cardiac and skeletal muscle cannot move quite as far without being impeded by actin-attached heads. “What leads to these differences?” Cremo wonders. “Are there differences in the biophysical behavior of the S2 domain in different muscle types?”     

A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

Cotton Rat Placenta Anatomy and Fc Receptor Expression and Their Roles in Maternal Antibody Transfer

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral pneumonia in infants and young children worldwide. Currently no vaccine is available to prevent RSV infection, but virus-neutralizing monoclonal antibodies can be given prophylactically, emphasizing the protective potential of antibodies. One concept of RSV vaccinology is mothers’ immunization to induce high antibody titers, leading to passive transfer of high levels of maternal antibody to the fetus through the placenta and to the neonate through colostrum. Cotton rats are an excellent small animal model for RSV infection and have been used to test maternal immunization. To mechanistically understand antibody transfer in the cotton rat model, we characterized the cotton rat placenta and Fc receptor localization. Placentas from cotton rats at midgestation (approximately day 14) and at late gestation (approximately day 25) and neonatal (younger than 1 wk) gastrointestinal tracts were collected for light microscopy, immunohistochemistry, and transmission electron microscopy. The cotton rat placenta is hemotrichorial and has 5 distinct layers: decidua, junctional zone, labyrinth, chorionic plate, and yolk sac. Consistent with the transfer of maternal antibodies, the majority of the Fc receptors are present in the yolk sac endoderm and fetal capillary endothelium of the chorionic plate, involving 10% of the cells within the labyrinth. In addition, Fc receptors are present on duodenal and jejunal enterocytes in cotton rats, similar to humans, mice, and rats. These findings provide the structural basis for the pre- and postnatal transfer of maternal antibodies described in cotton rats.

Cotton rats (Sigmodon hispidus) have been used to study the pathogenesis of human respiratory syncytial virus (RSV) infection.^6,15,29 This species is considered the best small animal model for RSV pathogenesis, because RSV replicates similarly in cotton rats as in infants and induces an adaptive immune response, as is seen in infants with moderately severe RSV infection.^4,5,33,38 Cotton rats have been used to accurately predict the efficacy of the only prophylactic treatment for RSV, a monoclonal antibody against the fusion protein.^5,17,31 In addition, cotton rats are the ‘gold standard’ for RSV vaccine development.^6,15,29,39A proposed method to protect infants from RSV-induced severe respiratory disease is to vaccinate pregnant mothers to increase the level of maternal antibodies transferred to fetuses and infants.² Human gestation has a duration of approximately 270 d, with midgestation corresponding to approximately day 135 and late gestation to approximately day 225. The human placenta is hemomonochorial in that trophoblasts are in direct contact with the maternal blood supply. A single continuous laver of trophoblasts separates fetal capillaries from the maternal blood.^23,34 Maternal antibodies are predominantly transferred in utero through the placenta. Maternal IgG is endocytosed by syncytiotrophoblasts, where IgG₁ and IgG₄ bind to the neonatal Fc receptor (FnRn) at an acidic pH in early endosomes.^11,25 The endosomal IgG-FcRn complex undergoes transcytosis and fuses with the basal membrane where IgG disassociates from the FcRn. The FcRn is then recycled back to the maternal blood surface of the syncytiotrophoblasts.^11,25,28,30 After birth, the FcRn in the intestine binds maternal IgG in the milk at the acidic apical surface of the epithelium and transports it in an endosome to the basolateral surface, where the maternal IgG disassociates and enters the neonatal blood stream.^20,21In cotton rats, gestation is 28 d in duration, with midgestation at approximately day 15 and late gestation at about day 25. The average litter size is 6 pups, with a range of 4 to 8 pups. Reportedly, maternal antibodies to RSV are transferred to cotton rat pups both prenatally and postnatally.³² Maternal immunization with RSV vaccine candidates in cotton rats has demonstrated that maternal antibodies are transferred to pups and confer protection, but this protection is only short-lived.² In contrast, maternal antibodies after measles immunization provide long-term protection in cotton rats.²⁸ Further investigation of the mechanisms of prenatal and postnatal antibody transfer in cotton rats is necessary to understand these differences and evaluate RSV vaccine candidates. As a basis for these physiologic studies, the placental anatomy and the expression of FcRn in cotton rat must be characterized. In this study, we report the histologic and ultrastructural characterization of the cotton rat placenta at mid- and late gestation and the localization of FcRn in the placenta and neonatal intestine.     

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague–Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 10⁶, 10⁷ or, 10⁸ CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 10⁷ or 10⁸ CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16.h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Protein binding is an important component of pharmacokinetic/pharmacodynamic (PK/PD) research. In vitro measurement of protein drug binding is an essential component of the research and development of novel drugs. However, in vitro studies often poorly mirror the in vivo condition.^9,42 Pharmacokinetic studies early in drug development provide a means to assess the time course of drug effects in the body and drug distribution and availability.⁴² From a PK/PD modeling perspective, protein binding is an important factor in the kinetics and dynamics of drug availability in vivo.^21,35,36,40 These complex relationships are used to project efficacious doses in humans and take into consideration differences in plasma protein binding between preclinical species and humans.^8,44A variety of acute phase proteins (APP) exist across all species and increase in response to inflammatory, infectious and traumatic events.^{5,9,12,13,19,21,22,29,45,53} APPs are potential biomarkers for detection and monitoring of various disease states including cancer.^{2,18,24,34,39,40,47,50,52} Because of this, enhanced understanding of drug binding characteristics to APPs early in the development phase will promote the design of more efficacious therapeutics. Alpha-1 acid glycoprotein (AGP), a ubiquitous major APP that is present in rats,^9,46 has significant drug binding properties and binds to many basic and neutral compounds. Normal AGP levels in plasma of naïve rats range from 0.1 to 0.32 mg/mL.⁴⁴ The importance of AGP as related to drug discovery and development will be bolstered by greater understanding of the sources of AGP stimulation in established animal models. For example, AGP modulates the immune response in a rodent shock model in which it is thought to maintain normal capillary permeability to ensure perfusion of vital organs.^30,33 In addition, elevated AGP levels are present in animal models of infection and inflammation.^{11,20,27,32,41,48}In surgically modified animals, AGP levels may be elevated after surgical manipulation, which unavoidably induces local transient inflammatory responses.^8,25,51 In addition, infections may develop postoperatively leading to increased AGP levels. Chronic catheterization has been linked to increased incidence of infection.^3,8,37 Surgically modified animals should not be placed on study if aseptic technique was not adhered to during surgical preparation and instrumentation.^6,37 Contamination may occur within or at the external portion of a catheter, usually resulting in more obvious signs of infection. Routine PK studies in rats involve implantation of vascular catheters through which drugs are administered and blood samples are taken over time. Catheterized animals are typically perceived as being healthy and thus are enrolled in and remain on study unless they develop obvious clinical signs of infection or illness. However, an occult infection may be present even with a patent catheter. As such, understanding the direct effect of subclinical infection in modulating AGP levels and drug binding is critical, as AGP levels may affect drug levels in study animals with persistent subclinical infection. In this event, the PK data generated may be altered due to selective binding to AGP, thus confounding data interpretation.A possible application of AGP is its potential utility as a biomarker for evaluating health status animals in drug development. The use of AGP as a select biomarker for monitoring and identifying sick animals and/or predicting the potential impact of subclinical infection on drug PK/PD is highly desirable. A screening tool such as this could help to optimize animal selection by reliably identifying healthy animals. Improved intra-study health monitoring would promote confidence in PK/PD data and its predictive value.The focus of this research was to develop a sensitive, reliable and reproducible model of subclinical infection in the rat using the ubiquitous skin contaminant, S. aureus. We selected AGP as a biomarker that would promote health status screening and enhance PK/PD characterization of AGP binding drugs (that is basic and neutral) in the presence or absence of subclinical infection. The model was validated by evaluating the impact of increased AGP levels on propranolol, a drug known to have high binding affinity to AGP.^{4,7,10,26,28,31,49} Ultimately, establishing this model will provide heightened visibility of the protein binding characteristics of drugs and yield more informed data interpretation.     

Fibrous Osteodystrophy,Chronic Renal Disease,and Uterine Adenocarcinoma in Aged Gray Mouse Lemurs (Microcebus murinus)

The gray mouse lemur (Microcebus murinus, GML) is a nocturnal, arboreal, prosimian primate that is native to Madagascar. Captive breeding colonies of GMLs have been established primarily for noninvasive studies on questions related to circadian rhythms and metabolism. GMLs are increasingly considered to be a strong translational model for neurocognitive aging due to overlapping histopathologic features shared with aged humans. However, little information is available describing the clinical presentations, naturally occurring diseases, and histopathology of aged GMLs. In our colony, a 9 y-old, male, GML was euthanized after sudden onset of weakness, lethargy, and tibial fracture. Evaluation of this animal revealed widespread fibrous osteodystrophy (FOD) of the mandible, maxilla, cranium, appendicular, and vertebral bones. FOD and systemic metastatic mineralization were attributed to underlying chronic renal disease. Findings in this GML prompted periodic colony-wide serum biochemical screenings for azotemia and electrolyte abnormalities. Subsequently, 3 additional GMLs (2 females and 1 male) were euthanized due to varying clinical and serum biochemical presentations. Common to all 4 animals were FOD, chronic renal disease, uterine adenocarcinoma (females only), cataracts, and osteoarthritis. This case study highlights the concurrent clinical and histopathologic abnormalities that are relevant to use of GMLs in the expanding field of aging research.

Within the past 5 y, recognition of the translational utility of the gray mouse lemur (Microcebus murinus, GML) has greatly expanded, in part due to the sequencing of its genome.²⁷ GMLs have been proposed as an animal model in the context of aging research,^14,35 most notably within the fields of Alzheimer disease and dementia^33,39 and circadian rhythms.^15,20 GMLs are nocturnal, arboreal, prosimian primates (family Cheirogaleidae) that are endemic to Madagascar. They are among the smallest primates, with a body weight of 49 to 80 g in the wild³⁷ (60 to 110 g in captivity) and have a life expectancy of approximately 8 to 10 y in captivity.¹⁴ A small number of captive breeding colonies have been established throughout Europe and the United States, many of which have arisen from a closed captive breeding colony at the Muséum National d''Histoire Naturelle (MNHN) in Brunoy, France.Despite an ever-growing interest in the GML as a model organism, clinical and pathologic case reports focusing on naturally occurring disease are rare for this species.^{1,4,10,16,17,20,28,31,34,38} Reports of spontaneous disease often focus on neoplasia^28,31,34 or on ocular abnormalities, which are accessible without invasive interventions.^1,4,12 Apart from age-related neurodegenerative disease and cognitive impairment,^{5,23,25,26,32,36} little is known about the natural disease predilection and histologic aging phenotypes of GMLs.In June 2017, a 9 y-old male GML was euthanized after the sudden onset of weakness, lethargy, and tibial fracture. Necropsy and histopathology revealed chronic renal disease, widespread fibrous osteodystrophy (FOD), and systemic metastatic mineralization. These findings prompted colony-wide serum biochemical screenings for potential underlying renal disease and subsequent metabolic bone disease within the population.Herein, we report the clinical, gross, and histologic multisystemic pathology of 4 aged GMLs. This is the first documentation of FOD secondary to chronic renal disease in GMLs in a captive research colony. In addition, we corroborate previous reports^31,34 of uterine adenocarcinoma in aged female GMLs. Together, these findings aid in providing appropriate clinical care to GMLs as their use in the field of aging research continues to expand.