Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

Interactions between pathogens cooccurring within a single host can have profound effects on infection outcomes, ranging from the severity of clinical disease in individual hosts to the rate of disease spread across populations (1–3). Because most hosts are commonly infected by more than one type of pathogen at a time (4), understanding the consequences of pathogen interactions during concurrent infection (or coinfection) is essential for effective disease management and control. While many studies focus on pathogen interactions that are the result of one pathogen responding to the simultaneous presence of another (5), two pathogens need not overlap in time to interact with one another. For example, heterologous immunity, where prior exposure or infection with one pathogen modifies the immune response to another, can drive both positive and negative interactions between pathogens (6). This phenomenon highlights how modifications of the host immune system by one pathogen that occur during the lifetime of a host (i.e., in ecological time) can shape future responses to secondary pathogens. Likewise, strong selection pressure imposed by pathogens on hosts, particularly on immune function (7), can result in modifications of the host immune system that occur over generations (i.e., in evolutionary time), a process which should also affect responses to secondary infections. In this case, a historical population-level response to selection by one pathogen may generate heritable differences among individuals in contemporary responses to another. Crucially, ecological- vs. evolutionary-scale interactions between pathogens may operate for different reasons, so distinguishing between the two is integral to understanding both the mechanistic basis and consequences of these interactions.Helminths, or parasitic worms, and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions (2, 8). Both pathogens affect approximately one-third of the world’s human population and are widespread in domestic and wild animals (9–11). Caused by bacteria in the Mycobacterium tuberculosis complex, including M. tuberculosis (Mtb), the causative agent of human TB, and Mycobacterium bovis (Mb), the causative agent of bovine TB, TB is responsible for 2 million human deaths (12) and 25% of all disease-related cattle deaths (13) annually. In humans, about 10% of individuals infected with Mtb progress to active pulmonary disease, but the mechanisms underlying progression to active TB are poorly defined (14). Accumulating evidence suggests that coinfection with worms may be a factor in TB disease progression (2, 15), although some studies do not support this link, highlighting the complex nature of worm–TB interactions (16). Interestingly, research in laboratory animals suggests that enhanced immunity (i.e., resistance) to worms can compromise a host’s ability to control TB even in the absence of active worm infection (17–20), implying that evolved defenses against worms may independently affect the response to TB. Considered in light of widespread worm resistance in human and animal populations (21, 22) and the broad geographic coincidence of worms and TB, worm–TB interactions may represent an illustrative case where variation in evolved resistance to one pathogen (worms) contributes to variable responses to another (TB).In this study, we tested the hypothesis that resistance to worms modifies the host response to TB. To do this, we monitored gastrointestinal (GI) worm (specifically strongyle nematode) and Mb infections in a cohort of wild African buffalo (Syncerus caffer) to assess the effects of natural variation in worm resistance on the incidence, severity, and progression of bovine TB (BTB). In previous work, we demonstrated the presence of an ecological interaction between worms and BTB in buffalo by showing that clearance of active worm infection via anthelmintic treatment reduces BTB-associated mortality (23). Thus, we took advantage of the fact that half of our study animals were subject to long-term deworming to compare the relative effects of worm coinfection vs. natural worm resistance on BTB outcomes. We found evidence of a genetic basis to worm resistance in buffalo and that buffalo with resistance to worms were more severely affected by BTB in terms of both mortality risk and disease progression. However, the mechanisms by which natural variation in the host response to worms was associated with BTB progression appeared to be distinct from the effects of anthelmintic treatment. Our results suggest that negative effects of worms on BTB outcomes occur as a result of both concurrent worm infection and genetically based differences in host responsiveness to worms. This discovery fundamentally alters our understanding of the timescales over which worms and TB interact in real-world populations.     

ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Loss-of-function mutations in ATP13A2 (PARK9) are causative for a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome (KRS, a juvenile onset parkinsonism with dementia) (1), early-onset Parkinson’s disease (PD) (2, 3), hereditary spastic paraplegia (HSP) (4), neuronal ceroid lipofuscinosis (5), and amyotrophic lateral sclerosis (6), which are commonly hallmarked by lysosomal and mitochondrial dysfunction (4, 6, 7). Also, ATP13A2 deficiency causes lysosomal and mitochondrial impairment in various models, as evidenced by decreased lysosomal functionality (8, 9), reduced mitochondrial clearance capacity (8–10), mitochondrial fragmentation, mitochondrial DNA damage, and increased oxygen consumption (11, 12).We recently discovered that ATP13A2 transports the polyamines spermidine and spermine from the late endo/lysosome to the cytosol (9). Polyamines are ubiquitous polycationic aliphatic amines that stabilize nucleic acids, influence protein folding, regulate ion channels, and modulate cell proliferation and differentiation (13–15). We found that the late endo-lysosomal transporter ATP13A2 strongly contributes to the total cellular polyamine content via a two-step process: Firstly, polyamines enter the cell via endocytosis and subsequently, polyamines are transported by ATP13A2 into the cytosol (9). This process complements polyamine biosynthesis via the ornithine decarboxylase (ODC) pathway (9). Importantly, ATP13A2’s polyamine transport function is crucial for its neuroprotective effect, since it prevents lysosomal polyamine accumulation and subsequent lysosomal rupture, while improving lysosomal health and functionality (9). Moreover, when activated by its two regulatory lipids—phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] and phosphatidic acid (PA)—ATP13A2 exerts a cell protective effect against the mitochondrial neurotoxin rotenone (16), an environmental risk factor for PD (17). Rotenone is a mitochondrial complex I inhibitor, which leads to high levels of reactive oxygen species (ROS), promoting protein aggregation and damaging organelles. However, how ATP13A2’s polyamine transport function exerts a cell protective effect against rotenone, or other mitochondrial neurotoxins, is not yet clear.Interestingly, the transported substrates spermine and spermidine reduce oxidative stress (14, 15). Spermine is a potent free radical scavenger (18) and a biologically important antioxidant (19–23). We therefore hypothesize that ATP13A2-mediated polyamine transport may counteract oxidative stress (16, 24) and preserve mitochondrial health (11, 12). Here, we demonstrate in complementary human cell models and Caenorhabditis elegans that lysosomal polyamine export by ATP13A2 effectively lowers ROS levels and promotes mitochondrial health and functionality, pointing to a lysosomal-dependent cell protective pathway that may be implicated in ATP13A2-related neurodegenerative disorders.     

Amputation-free survival in 17,353 people at high risk for foot ulceration in diabetes: a national observational study

Aims/hypothesis

Our aim was to investigate amputation-free survival in people at high risk for foot ulceration in diabetes (‘high-risk foot’), and to compare different subcategories of high-risk foot.

Methods

Overall, 17,353 people with diabetes and high-risk foot from January 2008 to December 2011 were identified from the Scotland-wide diabetes register (Scottish Care Information-Diabetes: N?=?247,278). Participants were followed-up for up to 2 years from baseline and were categorised into three groups: (1) those with no previous ulcer, (2) those with an active ulcer or (3) those with a healed previous ulcer. Participants with prior minor or major amputation were excluded. Accelerated failure time models were used to compare amputation-free survival up to 2 years between the three exposure groups.

Results

The 2 year amputation-free survival rate in all people with diabetes with high-risk foot was 84.5%. In this study group, 270 people (10.0%) had an amputation and 2424 (90.0%) died during the 2 year follow-up period. People who had active and healed previous ulcers at baseline had significantly lower 2 year amputation-free survival compared with those who had no previous ulcer (both p?<?0.0001). The percentage of people who died within 2 years for those with healed ulcer, active ulcer or no baseline ulcer was 22.8%, 16% and 12.1%, respectively.

Conclusions/interpretation

In people judged to be at high risk of foot ulceration, the risk of death was up to nine times the risk of amputation. Death rates were higher for people with diabetes who had healed ulcers than for those with active ulcers. However, people with active ulcers had the highest risk of amputation.

     

Gene control of tyrosine kinase TIE2 and vascular manifestations of infections

Ligands of the endothelial-enriched tunica interna endothelial cell kinase 2 (Tie2) are markedly imbalanced in severe infections associated with vascular leakage, yet regulation of the receptor itself has been understudied in this context. Here, we show that TIE2 gene expression may constitute a novel vascular barrier control mechanism in diverse infections. Tie2 expression declined rapidly in wide-ranging models of leak-associated infections, including anthrax, influenza, malaria, and sepsis. Forced Tie2 suppression sufficed to attenuate barrier function and sensitize endothelium to permeability mediators. Rapid reduction of pulmonary Tie2 in otherwise healthy animals attenuated downstream kinase signaling to the barrier effector vascular endothelial (VE)-cadherin and induced vascular leakage. Compared with wild-type littermates, mice possessing one allele of Tie2 suffered more severe vascular leakage and higher mortality in two different sepsis models. Common genetic variants that influence TIE2 expression were then sought in the HapMap3 cohort. Remarkably, each of the three strongest predicted cis-acting SNPs in HapMap3 was also associated with the risk of acute respiratory distress syndrome (ARDS) in an intensive care unit cohort of 1,614 subjects. The haplotype associated with the highest TIE2 expression conferred a 28% reduction in the risk of ARDS independent of other major clinical variables, including disease severity. In contrast, the most common haplotype was associated with both the lowest TIE2 expression and 31% higher ARDS risk. Together, the results implicate common genetic variation at the TIE2 locus as a determinant of vascular leak-related clinical outcomes from common infections, suggesting new tools to identify individuals at unusual risk for deleterious complications of infection.Among vascular-enriched receptor tyrosine kinases, Tie2 is unusual in at least two functional aspects. First, Tie2 phosphorylation is tightly controlled by the interplay of several proteins: a paralogous receptor, Tie1; a tyrosine phosphatase, vascular endothelial-protein tyrosine phosphatase (VE-PTP); and two secreted ligands, angiopoietin (Angpt)-1 and Angpt-2, the latter of which can act as an agonist, partial agonist, or antagonist depending upon context (1–6). Second, unlike classic growth factor receptors, Tie2 is heavily expressed and phosphorylated throughout the quiescent adult vasculature (7), suggesting that Tie2 signaling has one or more roles in vascular maintenance.Based largely on Angpt-1 overexpression studies, Tie2 has been implicated in vascular barrier defense (8, 9). However, adult-specific deletion of Angpt-1 does not appear to trigger vascular leakage (10). Moreover, Angpt-1 has repeatedly been ascribed functions that are independent of Tie2 (11–13). Finally, observational studies in humans suffering clinical manifestations of vascular leakage have consistently shown a marked imbalance in Tie2 ligands tilting in favor of Angpt-2 (reviewed in 14). Although decreased Tie2 activity has been inferred from these reports, the role of TIE2 gene expression has not been directly queried experimentally or in clinical settings.This question is important not only for understanding control mechanisms of the circulatory system but also to guide the development of strategies to predict, stratify, and treat patients affected by acute vascular leakage. If tonic Tie2 signaling is indeed necessary for vascular barrier maintenance, then reducing the pool of receptors could constitute a ligand-independent means to attenuate barrier-protective signaling in the endothelium. We therefore hypothesized that the level of Tie2 expression modulates the sensitivity of blood vessels, and thereby the entire organism, to noxious stimuli. Cellular, rodent, and human genetics studies were undertaken to test this concept.     

Cingulo-opercular control network and disused motor circuits joined in standby mode

Whole-brain resting-state functional MRI (rs-fMRI) during 2 wk of upper-limb casting revealed that disused motor regions became more strongly connected to the cingulo-opercular network (CON), an executive control network that includes regions of the dorsal anterior cingulate cortex (dACC) and insula. Disuse-driven increases in functional connectivity (FC) were specific to the CON and somatomotor networks and did not involve any other networks, such as the salience, frontoparietal, or default mode networks. Censoring and modeling analyses showed that FC increases during casting were mediated by large, spontaneous activity pulses that appeared in the disused motor regions and CON control regions. During limb constraint, disused motor circuits appear to enter a standby mode characterized by spontaneous activity pulses and strengthened connectivity to CON executive control regions.

Disuse is a powerful paradigm for inducing plasticity that has uncovered key organizing principles of the human brain (1–4). Monocular deprivation—prolonged covering of one eye—revealed that multiple afferent inputs can compete for representational territory in the primary visual cortex (1). Similar competition between afferents also shapes the somatomotor system. Manipulations such as peripheral nerve deafferentation, whisker trimming, and limb constraint all drive plasticity in the primary somatosensory and motor cortex (2–4). Most plasticity studies to date have used focal techniques, such as microelectrode recordings, to study local changes in brain function. As a result, little is known about how behavior and experience shape the brain-wide functional networks that support complex cognitive operations (5).The brain is composed of networks of regions that cooperate to perform specific cognitive functions (5–8). These functional networks show synchronized spontaneous activity while the brain is at rest, a phenomenon known as resting-state functional connectivity (FC) (9–11). FC can be measured noninvasively in humans using resting-state functional MRI (rs-fMRI) and has been used to parse the brain into canonical functional networks (12, 13), including visual, auditory, and somatomotor networks (14, 15); ventral and dorsal attention networks (8, 16); a default mode network with roles in internally directed cognition and episodic memory (7, 11); a salience network thought to assess the homeostatic relevance of external stimuli (17); a frontoparietal control network supporting error processing and moment-to-moment adjustments in behavior (18–20); and a cingulo-opercular control network (CON), which maintains executive control during goal-directed behavior (18, 19, 21). Each functional network likely carries out a variety of additional functions.A more recent advance in human neuroscience has been the recognition of individual variability in network organization (22–25). Most early rs-fMRI studies examined central tendencies in network organization using group-averaged FC measurements (10, 12, 13). Recent work has demonstrated that functional networks can be identified in an individual-specific manner if sufficient rs-fMRI data are acquired, an approach termed precision functional mapping (PFM) (22, 23, 26–30). PFM respects the unique functional anatomy of each person and avoids averaging together functionally distinct brain regions across individuals.We recently demonstrated that PFM can be used to follow the time course of disuse-driven plasticity in the human brain (31). Three adult participants (Nico, Ashley, and Omar) were scanned at the same time of day for 42 to 64 consecutive days (30 min of rs-fMRI per day) before, during, and after 2 wk of dominant upper-extremity casting (Fig. 1 A and B). Casting caused persistent disuse of the dominant upper extremity during daily behaviors and led to a marked loss of strength and fine motor skill in all participants. During casting, the upper-extremity regions of the left primary somatomotor cortex (L-SM1_ue) and right cerebellum (R-Cblm_ue) functionally disconnected from the remainder of the somatomotor network. Disused motor circuits also exhibited large, spontaneous pulses of activity (Fig. 1C). Disuse pulses did not occur prior to casting, started to occur frequently within 1 to 2 d of casting, and quickly waned after cast removal.Open in a separate windowFig. 1.Experimental design and spontaneous activity pulses. (A) Three participants (Nico, Ashley, and Omar) wore casts covering the entire dominant upper extremity for 2 wk. (B) Participants were scanned every day for 42 to 64 consecutive days before, during, and after casting. All scans included 30 min of resting-state functional MRI. (C) During the Cast period, disused somatomotor circuits exhibited large pulses of spontaneous activity. (C, Left) Whole-brain ANOVA showing which brain regions contained disuse-driven pulses. (C, Right) Time courses of all pulses recorded from the disused primary somatomotor cortex.Somatomotor circuits do not function in isolation. Action selection and motor control are thought to be governed by complex interactions between the somatomotor network and control networks, including the CON (18). Prior studies of disuse-driven plasticity, including our own, have focused solely on somatomotor circuits. Here, we leveraged the whole-brain coverage of rs-fMRI and the statistical power of PFM to examine disuse-driven plasticity throughout the human brain.     

Changing nutrient cycling in Lake Baikal,the world’s oldest lake

Lake Baikal, lying in a rift zone in southeastern Siberia, is the world''s oldest, deepest, and most voluminous lake that began to form over 30 million years ago. Cited as the “most outstanding example of a freshwater ecosystem” and designated a World Heritage Site in 1996 due to its high level of endemicity, the lake and its ecosystem have become increasingly threatened by both climate change and anthropogenic disturbance. Here, we present a record of nutrient cycling in the lake, derived from the silicon isotope composition of diatoms, which dominate aquatic primary productivity. Using historical records from the region, we assess the extent to which natural and anthropogenic factors have altered biogeochemical cycling in the lake over the last 2,000 y. We show that rates of nutrient supply from deep waters to the photic zone have dramatically increased since the mid-19th century in response to changing wind dynamics, reduced ice cover, and their associated impact on limnological processes in the lake. With stressors linked to untreated sewage and catchment development also now impacting the near-shore region of Lake Baikal, the resilience of the lake’s highly endemic ecosystem to ongoing and future disturbance is increasingly uncertain.

Ancient lakes have long been associated with both high levels of biodiversity and endemicity. However, they are also being threatened by anthropogenic forcings that have led to impacts ranging from the warming of lake waters (1), hydrological modifications (2), increases in aquatic toxicity (3), and declining endemic populations due to introductions of nonnative species (4). With global populations increasingly reliant on large and ancient lakes for ecosystem services, the biodiversity (5) and value of aquatic systems to society (6), particularly in ancient lake systems (7), are at risk. Lake Baikal (Russia) is an exceptional example of an ancient lake (Fig. 1). In addition to containing ∼20% of global surface freshwater, the lake is characterized by its high degree of biodiversity with over 2,500 flora and fauna, the majority of which are endemic (8). This has been attributed to the lake’s age and fully oxygenated water column, driven by seasonal overturning and deep water renewal (9, 10) that sustains an almost completely endemic deep water fauna (8).Open in a separate windowFig. 1.Location of Lake Baikal and its catchment (gray) together with the location of World Meteorological Organization station in Irkutsk, major catchment rivers (brown), coring sites (BAIK13-1, BAIK13-4), and sites providing additional data used in this study (BAIK13-7).Concerns exist over the future health of this unique ecosystem, amid evidence of extensive shoreline eutrophication (11, 12) and climate-induced shifts in primary productivity (13, 14). Together, these changes have impacted organisms ranging from sponges and gastropods to ciliates, flagellates, and algal communities (15). Given the likelihood of future anthropogenic disturbance on Lake Baikal, further disrupting productivity exchanges through the lake’s food web, there is a need to place these contemporary observations into their historical setting. In Lake Baikal, we have evidence that algal communities have undergone rapid multidecadal to multicentennial timescale changes over the last 2,000 y (16). However, there is a need to also gain a clearer insight into how biogeochemical and nutrient cycling has altered over the same timescale, both to contextualize natural and anthropogenic drivers of change and to understand the susceptibility of the lake’s ecosystem to further alteration under different climate states (17). Annual primary productivity in Lake Baikal is ultimately regulated by photic zone nutrient availability, in addition to ice/snow cover, which regulates light availability for photosynthesis (10, 18). Here, by analyzing the silicon isotope composition of diatom silica (δ³⁰Si_diatom), we show that nutrient supply to the surface waters of Lake Baikal has rapidly increased through the 20th and 21st centuries coincident with increased wind-driven Ekman transport and reduced ice cover. These changes in photic zone nutrient availability have the potential to alter resource competition and prey–predator interactions across the lake (15, 19).     

Fluoroquinolone Resistance Does Not Impose a Cost on the Fitness of Clostridium difficile In Vitro

Point mutations conferring resistance to fluoroquinolones were introduced in the gyr genes of the reference strain Clostridium difficile 630. Only mutants with the substitution Thr-82→Ile in GyrA, which characterizes the hypervirulent epidemic clone III/027/NAP1, were resistant to all fluoroquinolones tested. The absence of a fitness cost in vitro for the most frequent mutations detected in resistant clinical isolates suggests that resistance will be maintained even in the absence of antibiotic pressure.