Parasite Survival and Disease Persistence in Cystic Fibrosis, Schistosomiasis and Pathogenic Bacterial Diseases: A Role for Universal Stress Proteins?

Universal stress proteins (USPs) were originally discovered in Escherichia coli over two decades ago and since then their presence has been detected in various organisms that include plants, archaea, metazoans, and bacteria. As their name suggests, they function in a series of various cellular responses in both abiotic and biotic stressful conditions such as oxidative stress, exposure to DNA damaging agents, nutrient starvation, high temperature and acidic stress, among others. Although a highly conserved group of proteins, the molecular and biochemical aspects of their functions are largely evasive. This is concerning, as it was observed that USPs act as essential contributors to the survival/persistence of various infectious pathogens. Their ubiquitous nature in various organisms, as well as their augmentation during conditions of stress, is a clear indication of their direct or indirect importance in providing resilience against such conditions. This paper seeks to clarify what has already been reported in the literature on the proposed mechanism of action of USPs in pathogenic organisms.

Understanding the Pathophysiology of Exosomes in Schistosomiasis: A New Direction for Disease Control and Prevention.

Schistosomiasis is a parasitic disease endemic to freshwater areas of Southeast Asia, Africa, and South America that is capable of causing serious damage to the internal organs. Recent studies have linked exosomes to the progression of schistosomiasis. These structures are important mediators for intercellular communication, assist cells to exchange proteins, lipids, and genetic material and have been shown to play critical roles during host-parasite interactions. This review aims to discuss the pathophysiology of exosomes in schistosomiasis and their roles in regulating the host immune response. Understanding how exosomes are involved in the pathogenesis of schistosomiasis may provide new perspectives in diagnosing and treating this neglected disease.

Immunological and Biochemical Interplay between Cytokines, Oxidative Stress and Schistosomiasis.

The host-parasite schistosome relationship relies heavily on the interplay between the strategies imposed by the schistosome worm and the defense mechanisms the host uses to counter the line of attack of the parasite. The ultimate goal of the schistosome parasite entails five important steps: evade elimination tactics, survive within the human host, develop into adult forms, propagate in large numbers, and transmit from one host to the next. The aim of the parasitized host on the other hand is either to cure or limit infection. Therefore, it is a battle between two conflicting aspirations. From the host's standpoint, infection accompanies a plethora of immunological consequences; some are set in place to defend the host, while most end up promoting chronic disease, which ultimately crosses paths with oxidative stress and cancer. Understanding these networks provides attractive opportunities for anti-schistosome therapeutic development. Hence, this review discusses the mechanisms by which schistosomes modulate the human immune response with ultimate links to oxidative stress and genetic instability.

Schistosomes in the Lung: Immunobiology and Opportunity.

Schistosome infection is a major cause of global morbidity, particularly in sub-Saharan Africa. However, there is no effective vaccine for this major neglected tropical disease, and re-infection routinely occurs after chemotherapeutic treatment. Following invasion through the skin, larval schistosomula enter the circulatory system and migrate through the lung before maturing to adulthood in the mesenteric or urogenital vasculature. Eggs released from adult worms can become trapped in various tissues, with resultant inflammatory responses leading to hepato-splenic, intestinal, or urogenital disease - processes that have been extensively studied in recent years. In contrast, although lung pathology can occur in both the acute and chronic phases of schistosomiasis, the mechanisms underlying pulmonary disease are particularly poorly understood. In chronic infection, egg-mediated fibrosis and vascular destruction can lead to the formation of portosystemic shunts through which eggs can embolise to the lungs, where they can trigger granulomatous disease. Acute schistosomiasis, or Katayama syndrome, which is primarily evident in non-endemic individuals, occurs during pulmonary larval migration, maturation, and initial egg-production, often involving fever and a cough with an accompanying immune cell infiltrate into the lung. Importantly, lung migrating larvae are not just a cause of inflammation and pathology but are a key target for future vaccine design. However, vaccine efforts are hindered by a limited understanding of what constitutes a protective immune response to larvae. In this review, we explore the current understanding of pulmonary immune responses and inflammatory pathology in schistosomiasis, highlighting important unanswered questions and areas for future research.

Identification of Low Molecular Weight Proteins and Peptides from Schistosoma mekongi Worm, Egg and Infected Mouse Sera.

Schistosoma mekongi is found in the lower Mekong river region and causes schistosomiasis. Low sensitivity of diagnosis and development of drug resistance are problems to eliminate this disease. To develop novel therapies and diagnostics for S. mekongi, the basic molecular biology of this pathogen needs to be explored. Bioactive peptides have been reported in several worms and play important roles in biological functions. Limited information is available on the S. mekongi peptidome. Therefore, this study aimed to identify S. mekongi peptides using in silico transcriptome mining and mass spectrometry approaches. Schistosoma peptide components were identified in adult worms, eggs, and infected mouse sera. Thirteen neuropeptide families were identified using in silico predictions from in-house transcriptomic databases of adult S. mekongi worms. Using mass spectrometry approaches, 118 peptides (from 54 precursor proteins) and 194 peptides (from 86 precursor proteins) were identified from adult worms and eggs, respectively. Importantly, eight unique peptides of the S. mekongi ubiquitin thioesterase, trabid, were identified in infected mouse sera 14, 28, and 56 days after infection. This protein may be a potential target for diagnosis of schistosomiasis. The S. mekongi peptide profiles determined in this study could be used for further drug and diagnostic development.

Immunological Considerations for Schistosoma Vaccine Development: Transitioning to Endemic Settings.

Despite mass drug administration programmes with praziquantel, the prevalence of schistosomiasis remains high. A vaccine is urgently needed to control transmission of this debilitating disease. As some promising schistosomiasis vaccine candidates are moving through pre-clinical and clinical testing, we review the immunological challenges that these vaccine candidates may encounter in transitioning through the clinical trial phases in endemic settings. Prior exposure of the target population to schistosomes and other infections may impact vaccine response and efficacy and therefore requires considerable attention. Schistosomes are known for their potential to induce T-reg/IL-10 mediated immune suppression in populations which are chronically infected. Moreover, endemicity of schistosomiasis is focal whereby target and trial populations may exhibit several degrees of prior exposure as well as in utero exposure which may increase heterogeneity of vaccine responses. The age dependent distribution of exposure and development of acquired immunity, and general differences in the baseline immunological profile, adds to the complexity of selecting suitable trial populations. Similarly, prior or concurrent infections with other parasitic helminths, viral and bacterial infections, may alter immunological responses. Consequently, treatment of co-infections may benefit the immunogenicity of vaccines and may be considered despite logistical challenges. On the other hand, viral infections leave a life-long immunological imprint on the human host. Screening for serostatus may be needed to facilitate interpretation of vaccine responses. Co-delivery of schistosome vaccines with PZQ is attractive from a perspective of implementation but may complicate the immunogenicity of schistosomiasis vaccines. Several studies have reported PZQ treatment to induce both transient and long-term immuno-modulatory effects as a result of tegument destruction, worm killing and subsequent exposure of worm antigens to the host immune system. These in turn may augment or antagonize vaccine immunogenicity. Understanding the complex immunological interactions between vaccine, co-infections or prior exposure is essential in early stages of clinical development to facilitate phase 3 clinical trial design and implementation policies. Besides well-designed studies in different target populations using schistosome candidate vaccines or other vaccines as models, controlled human infections could also help identify markers of immune protection in populations with different disease and immunological backgrounds.

Arachidonic Acid Is a Safe and Efficacious Schistosomicide, and an Endoschistosomicide in Natural and Experimental Infections, and Cysteine Peptidase Vaccinated Hosts.

Blood flukes of the genus Schistosoma are covered by a protective heptalaminated, double lipid bilayer surface membrane. Large amounts of sphingomyelin (SM) in the outer leaflet form with surrounding water molecules a tight hydrogen bond barrier, which allows entry of nutrients and prevents access of host immune effectors. Excessive hydrolysis of SM to phosphoryl choline and ceramide via activation of the parasite tegument-associated neutral sphingomyelinase (nSMase) with the polyunsaturated fatty acid, arachidonic acid (ARA) leads to parasite death, via allowing exposure of apical membrane antigens to antibody-dependent cell-mediated cytotoxicity (ADCC), and accumulation of the pro-apoptotic ceramide. Surface membrane nSMase represents, thus, a worm Achilles heel, and ARA a valid schistosomicide. Several experiments conducted in vitro using larval, juvenile, and adult Schistosoma mansoni and Schistosoma haematobium documented ARA schistosomicidal potential. Arachidonic acid schistosomicidal action was shown to be safe and efficacious in mice and hamsters infected with S. mansoni and S. haematobium, respectively, and in children with light S. mansoni infection. A combination of praziquantel and ARA led to outstanding cure rates in children with heavy S. mansoni infection. Additionally, ample evidence was obtained for the powerful ARA ovocidal potential in vivo and in vitro against S. mansoni and S. haematobium liver and intestine eggs. Studies documented ARA as an endogenous schistosomicide in the final mammalian and intermediate snail hosts, and in mice and hamsters, immunized with the cysteine peptidase-based vaccine. These findings together support our advocating the nutrient ARA as the safe and efficacious schistosomicide of the future.

A systematic review and meta-analysis on the rate of human schistosomiasis reinfection.

While praziquantel mass drug administration is currently the most widely used method in the control of human schistosomiasis, it does not prevent subsequent reinfection hence persistent transmission. Towards schistosomiasis elimination, understanding the reinfection rate is crucial in planning for the future interventions. However, there is scarcity of information on the global reinfection rate of schistosomiasis. This systematic review and meta-analysis aimed at summarizing studies that estimated the reinfection rate of human schistosomiasis. Three data bases (PubMed, Hinari and Google Scholar) were thoroughly searched to retrieve original research articles presenting data on reinfection rate of human schistosomiasis. Study quality and risk of bias was assessed based on Joanna Briggs Institute critical appraisal checklist. Meta-analysis was conducted using statistical R version 3.6.2 and R Studio using "meta" and "metafor" packages. Random effect model was employed to estimate pooled reinfection rates. Heterogeneity was determined using Cochran's Q (chi-square)-test and Higgins I2 statistics. A total of 29 studies met inclusion criteria to be included in this review. All studies had at least satisfactory (5-9 scores) quality. The overal mean and pooled reinfection rates of schistosomiasis were 36.1% (±23.3%) and 33.2% (95% CI, 26.5-40.5%) respectively. For intestinal schistosomiasis, the mean and pooled reinfection rates were 43.9% (±20.6%) and 43.4% (95% CI, 35.8-51.4%), and that for urogenital schistosomiasis were 17.6% (±10.8%) and 19.4% (95% CI, 12.3%- 29.2%) respectively. Cochran's Q (chi-square)-test and Higgins I2 statistic indicated significant heterogeneity across studies (p-values < 0.001, I2 values > 95%). Results of subgroup analysis showed that, the type of Schistosoma species, participants' age group, sample size and geographical area had influence on disparity variation in reinfection rate of schistosomiasis (p < 0.1). Despite the control measures in place, the re-infection rate is still high, specifically on intestinal schistosomiasis as compared to urogenital schistosomiasis. Achieving 2030 sustainable development goal 3 on good health and wellbeing intensive programmatic strategies for schistosomiasis elimination should be implemented. Among such strategies to be used at national level are repeated mass drug administration at least every six months, intensive snails control and health education.

Nitric oxide debilitates the neuropathogenic schistosome Trichobilharzia regenti in mice, partly by inhibiting its vital peptidases.

BACKGROUND: Avian schistosomes, the causative agents of human cercarial dermatitis (or swimmer's itch), die in mammals but the mechanisms responsible for parasite elimination are unknown. Here we examined the role of reactive nitrogen species, nitric oxide (NO) and peroxynitrite, in the immune response of mice experimentally infected with Trichobilharzia regenti, a model species of avian schistosomes remarkable for its neuropathogenicity. METHODS: Inducible NO synthase (iNOS) was localized by immunohistochemistry in the skin and the spinal cord of mice infected by T. regenti. The impact of iNOS inhibition by aminoguanidine on parasite burden and growth was then evaluated in vivo. The vulnerability of T. regenti schistosomula to NO and peroxynitrite was assessed in vitro by viability assays and electron microscopy. Additionally, the effect of NO on the activity of T. regenti peptidases was tested using a fluorogenic substrate. RESULTS: iNOS was detected around the parasites in the epidermis 8 h post-infection and also in the spinal cord 3 days post-infection (dpi). Inhibition of iNOS resulted in slower parasite growth 3 dpi, but the opposite effect was observed 7 dpi. At the latter time point, moderately increased parasite burden was also noticed in the spinal cord. In vitro, NO did not impair the parasites, but inhibited the activity of T. regenti cathepsins B1.1 and B2, the peptidases essential for parasite migration and digestion. Peroxynitrite severely damaged the surface tegument of the parasites and decreased their viability in vitro, but rather did not participate in parasite clearance in vivo. CONCLUSIONS: Reactive nitrogen species, specifically NO, do not directly kill T. regenti in mice. NO promotes the parasite growth soon after penetration (3 dpi), but prevents it later (7 dpi) when also suspends the parasite migration in the CNS. NO-related disruption of the parasite proteolytic machinery is partly responsible for this effect.

SCORE Studies on the Impact of Drug Treatment on Morbidity due to Schistosoma mansoni and Schistosoma haematobium Infection.

The Schistosomiasis Consortium for Operational Research (SCORE) was funded in 2008 to improve the evidence base for control and elimination of schistosomiasis-better understanding of the systemic morbidities experienced by children in schistosomiasis-endemic areas and the response of these morbidities to treatment, being essential for updating WHO guidelines for mass drug administration (MDA) in endemic areas. This article summarizes the SCORE studies that aimed to gauge the impact of MDA-based treatment on schistosomiasis-related morbidities. Morbidity cohort studies were embedded in the SCORE's larger field studies of gaining control of schistosomiasis in Kenya and Tanzania. Following MDA, cohort children had less undernutrition, less portal vein dilation, and increased quality of life in Year 5 compared with baseline. We also conducted a pilot study of the Behavioral Assessment System for Children (BASC-2) in conjunction with the Kenya gaining control study, which demonstrated beneficial effects of treatment on classroom behavior. In addition, the SCORE's Rapid Answers Project performed systematic reviews of previously available data, providing two meta-analyses related to morbidity. The first documented children's infection-related deficits in school attendance and achievement and in formal tests of learning and memory. The second showed that greater reductions in egg output following drug treatment correlates significantly with reduced odds of most morbidities. Overall, these SCORE morbidity studies provided convincing evidence to support the use of MDA to improve the health of school-aged children in endemic areas. However, study findings also support the need to use enhanced metrics to fully assess and better control schistosomiasis-associated morbidity.

Genetic algorithms identify individuals with high risk of severe liver disease caused by schistosomes.

Schistosomes induce severe hepatic disease, which is fatal in 2-10% of cases, mortality being higher in cases of co-infection with HBV or HCV. Hepatic disease occurs as a consequence of the chronic inflammation caused by schistosome eggs trapped in liver sinusoids. In certain individuals, the repair process leads to a massive accumulation of fibrosis in the periportal spaces. We and others have shown that genetic variants play a crucial role in disease progression from mild to severe fibrosis and explain why hepatic fibrosis progresses rapidly in certain subjects only. We will review here published findings concerning the strategies that have been used in the analysis of hepatic fibrosis in schistosome-infected individuals, the genetic variants that have associated with fibrosis, and variants in new pathways crucial for fibrosis progression. Together, these studies show that the development of fibrosis is under the tight genetic control of various common variants with moderate effects. This polygenic control has made it possible to develop models that identify schistosome-infected individual at risk of severe hepatic disease. We discuss the performances and limitations of these models.

Immune Evasion Strategies of Schistosomes.

Human schistosomes combat the unique immune systems of two vastly different hosts during their indirect life cycles. In gastropod molluscs, they face a potent innate immune response composed of variable immune recognition molecules and highly phagocytic hemocytes. In humans, a wide variety of innate and adaptive immune processes exist in proximity to these parasites throughout their lifespan. To survive and thrive as the second most common parasitic disease in humans, schistosomes have evolved many techniques to avoid and combat these targeted host responses. Among these techniques are molecular mimicry of host antigens, the utilization of an immune resistant outer tegument, the secretion of several potent proteases, and targeted release of specific immunomodulatory factors affecting immune cell functions. This review seeks to describe these key immune evasion mechanisms, among others, which schistosomes use to survive in both of their hosts. After diving into foundational observational studies of the processes mediating the establishment of schistosome infections, more recent transcriptomic and proteomic studies revealing crucial components of the host/parasite molecular interface are discussed. In order to combat this debilitating and lethal disease, a comprehensive understanding of schistosome immune evasion strategies is necessary for the development of novel therapeutics and treatment plans, necessitating the discussion of the numerous ways in which these parasitic flatworms overcome the immune responses of both hosts.

Why Do Intravascular Schistosomes Coat Themselves in Glycolytic Enzymes?

Schistosomes are intravascular parasitic helminths (blood flukes) that infect more than 200 million people globally. Proteomic analysis of the tegument (skin) of these worms has revealed the surprising presence of glycolytic enzymes on the parasite's external surface. Immunolocalization data as well as enzyme activity displayed by live worms confirm that functional glycolytic enzymes are indeed expressed at the host-parasite interface. Since these enzymes are traditionally considered to function intracellularly to drive glycolysis, in an extracellular location they are hypothesized to engage in novel "moonlighting" functions such as immune modulation and blood clot dissolution that promote parasite survival. For instance, several glycolytic enzymes can interact with plasminogen and promote its activation to the thrombolytic plasmin; some can inhibit complement function; some induce B cell proliferation or macrophage apoptosis. Several pathogenic bacteria and protists also express glycolytic enzymes externally, suggesting that moonlighting functions of extracellular glycolytic enzymes can contribute broadly to pathogen virulence. Also see the video abstract here https://youtu.be/njtWZ2y3k_I.

Crystal structure and chemical inhibition of essential schistosome host-interactive virulence factor carbonic anhydrase SmCA.

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Here we identify an α-carbonic anhydrase (SmCA) that is expressed at the schistosome surface as determined by activity assays and immunofluorescence/immunogold localization. Suppressing SmCA expression by RNAi significantly impairs the ability of larval parasites to infect mice, validating SmCA as a rational drug target. Purified, recombinant SmCA possesses extremely rapid CO2 hydration kinetics (kcat: 1.2 × 106 s-1; kcat/Km: 1.3 × 108 M-1s-1). The enzyme's crystal structure was determined at 1.75 Å resolution and a collection of sulfonamides and anions were tested for their ability to impede rSmCA action. Several compounds (phenylarsonic acid, phenylbaronic acid, sulfamide) exhibited favorable Kis for SmCA versus two human isoforms. Such selective rSmCA inhibitors could form the basis of urgently needed new drugs that block essential schistosome metabolism, blunt parasite virulence and debilitate these important global pathogens.

Intravital imaging of host-parasite interactions in skin and adipose tissues.

Intravital microscopy allows the visualisation of how pathogens interact with host cells and tissues in living animals in real time. This method has enabled key advances in our understanding of host-parasite interactions under physiological conditions. A combination of genetics, microscopy techniques, and image analysis have recently facilitated the understanding of biological phenomena in living animals at cellular and subcellular resolution. In this review, we summarise findings achieved by intravital microscopy of the skin and adipose tissues upon infection with various parasites, and we present a view into possible future applications of this method.

Genetic Crosses and Linkage Mapping in Schistosome Parasites.

Linkage mapping - utilizing experimental genetic crosses to examine cosegregation of phenotypic traits with genetic markers - is now 100 years old. Schistosome parasites are exquisitely well suited to linkage mapping approaches because genetic crosses can be conducted in the laboratory, thousands of progeny are produced, and elegant experimental work over the last 75 years has revealed heritable genetic variation in multiple biomedically important traits such as drug resistance, host specificity, and virulence. Application of this approach is timely because the improved genome assembly for Schistosoma mansoni and developing molecular toolkit for schistosomes increase our ability to link phenotype with genotype. We describe current progress and potential future directions of linkage mapping in schistosomes.