Fungal Spores in Insect Trapping Fluids: Simultaneous Sampling for Insects and Pathogens

Surveillance for early detection of non-native, invasive pathogens requires simple, sturdy, and easy to use collecting devices. In this study, we compared the fungal species detected in wet collection cups of Lindgren traps versus those detected on slides with oiled cheesecloth as aerial spore collectors. DNA was extracted and amplified from both using the primers ITS1F -ITS7G, and Illumina sequencing was used for metabarcoding of fungi present in samples. In 90 samples there were 1277 fungal OTUs. For fungal OTUs only detected by one collection method, insect traps had three times the number of fungal OTUs compared to slides, and this pattern persisted when analyses were restricted to pathogens and forest pathogens. Annually, thousands of insect traps are deployed in North America and the associated trap fluids have added value in forest disease research and monitoring.

Application of a Multiplex Polymerase Chain Reaction Test for Diagnosing Bacterial Enteritis in Children in a Real-Life Clinical Setting

This study aimed to determine the subjects for bacterial multiplex polymerase chain reaction (mPCR) testing and to interpret the mPCR test results based on patients’ clinical symptoms and diagnoses. The medical records of 710 pediatric patients who underwent a bacterial mPCR test were retrospectively reviewed. Clinical characteristics and mPCR test results were compared between patients with positive (n = 199) and negative mPCR test results (n = 511) and between patients with invasive pathogens (n = 95) and toxigenic pathogens (n = 70). Positive mPCR test results were significantly associated with older age (p < 0.001), diagnosis of acute gastroenteritis (p = 0.021), presence of hematochezia (p < 0.001), and absence of cough (p = 0.004). The diagnosis of acute gastroenteritis (p = 0.003), presence of fever (p = 0.027) and diarrhea (p = 0.043), and higher C-reactive protein levels (p = 0.025) were significantly associated with the identification of invasive pathogens in patients with positive mPCR test results. Thus, selective bacterial mPCR testing should be performed based on the patients’ clinical symptoms and diagnoses, and the results should be interpreted in consideration with identified pathogens.

Co-Adaptation of Physical Attributes of the Mammalian Female Reproductive Tract and Sperm to Facilitate Fertilization

The functions of the female reproductive tract not only encompass sperm migration, storage, and fertilization, but also support the transport and development of the fertilized egg through to the birth of offspring. Further, because the tract is open to the external environment, it must also provide protection against invasive pathogens. In biophysics, sperm are considered “pusher microswimmers”, because they are propelled by pushing fluid behind them. This type of swimming by motile microorganisms promotes the tendency to swim along walls and upstream in gentle fluid flows. Thus, the architecture of the walls of the female tract, and the gentle flows created by cilia, can guide sperm migration. The viscoelasticity of the fluids in the tract, such as mucus secretions, also promotes the cooperative swimming of sperm that can improve fertilization success; at the same time, the mucus can also impede the invasion of pathogens. This review is focused on how the mammalian female reproductive tract and sperm interact physically to facilitate the movement of sperm to the site of fertilization. Knowledge of female/sperm interactions can not only explain how the female tract can physically guide sperm to the fertilization site, but can also be applied for the improvement of in vitro fertilization devices.

Systems Biology Modeling of the Complement System Under Immune Susceptible Pathogens

The complement system is assembled from a network of proteins that function to bring about the first line of defense of the body against invading pathogens. However, complement deficiencies or invasive pathogens can hijack complement to subsequently increase susceptibility of the body to infections. Moreover, invasive pathogens are increasingly becoming resistant to the currently available therapies. Hence, it is important to gain insights into the highly dynamic interaction between complement and invading microbes in the frontlines of immunity. Here, we developed a mathematical model of the complement system composed of 670 ordinary differential equations with 328 kinetic parameters, which describes all three complement pathways (alternative, classical, and lectin) and includes description of mannose-binding lectin, collectins, ficolins, factor H-related proteins, immunoglobulin M, and pentraxins. Additionally, we incorporate two pathogens: (type 1) complement susceptible pathogen and (type 2) Neisseria meningitidis located in either nasopharynx or bloodstream. In both cases, we generate time profiles of the pathogen surface occupied by complement components and the membrane attack complex (MAC). Our model shows both pathogen types in bloodstream are saturated by complement proteins, whereas MACs occupy &lt;&lt;1.0% of the pathogen surface. Conversely, the MAC production in nasopharynx occupies about 1.5–10% of the total N. meningitidis surface, thus making nasal MAC levels at least about eight orders of magnitude higher. Altogether, we predict complement-imbalance, favoring overactivation, is associated with nasopharynx homeostasis. Conversely, orientating toward complement-balance may cause disruption to the nasopharynx homeostasis. Thus, for sporadic meningococcal disease, our model predicts rising nasal levels of complement regulators as early infection biomarkers.

Screening a Natural Product-Inspired Library for Anti-Phytophthora Activities

Phytophthora is a genus of microorganisms that cause devastating dieback and root-rot diseases in thousands of plant hosts worldwide. The economic impact of Phytophthora diseases on crops and native ecosystems is estimated to be billions of dollars per annum. These invasive pathogens are extremely difficult to control using existing chemical means, and the effectiveness of the few treatments available is being jeopardized by increasing rates of resistance. There is an urgent need to identify new chemical treatments that are effective against Phytophthora diseases. Natural products have long been regarded as “Nature’s medicine chest”, providing invaluable leads for developing front-line drugs and agrochemical agents. Here, we have screened a natural product-inspired library of 328 chemicals against two key Phytophthora species: Phytophthora cinnamomi and Phytophthora agathidicida. The library was initially screened for inhibition of zoospore germination. From these screens, we identified twenty-one hits that inhibited germination of one or both species. These hits were further tested in mycelial growth inhibition studies to determine their half-maximal inhibitory concentrations (IC50s). Four compounds had IC50 values of approximately 10 µM or less, and our best hit had IC50s of approximately 3 µM against both Phytophthora species tested. Overall, these hits may serve as promising leads for the development of new anti-Phytophthora agrochemicals

PKCα Is Recruited to Staphylococcus aureus-Containing Phagosomes and Impairs Bacterial Replication by Inhibition of Autophagy

Hijacking the autophagic machinery is a key mechanism through which invasive pathogens such as Staphylococcus aureus replicate in their host cells. We have previously demonstrated that the bacteria replicate in phagosomes labeled with the autophagic protein LC3, before escaping to the cytoplasm. Here, we show that the Ca2+-dependent PKCα binds to S. aureus-containing phagosomes and that α-hemolysin, secreted by S. aureus, promotes this recruitment of PKCα to phagosomal membranes. Interestingly, the presence of PKCα prevents the association of the autophagic protein LC3. Live cell imaging experiments using the PKC activity reporter CKAR reveal that treatment of cells with S. aureus culture supernatants containing staphylococcal secreted factors transiently activates PKC. Functional studies reveal that overexpression of PKCα causes a marked inhibition of bacterial replication. Taken together, our data identify enhancing PKCα activity as a potential approach to inhibit S. aureus replication in mammalian cells.

Taurine Alleviates Streptococcus uberis-Induced Inflammation by Activating Autophagy in Mammary Epithelial Cells

Streptococcus uberis infection can cause serious inflammation and damage to mammary epithelial cells and tissues that can be significantly alleviated by taurine. Autophagy plays an important role in regulating immunity and clearing invasive pathogens and may be regulated by taurine. However, the relationships between taurine, autophagy, and S. uberis infection remain unclear. Herein, we demonstrate that taurine augments PTEN activity and inhibits Akt/mTOR signaling, which decreases phosphorylation of ULK1 and ATG13 by mTOR and activates autophagy. Activating autophagy accelerates the degradation of intracellular S. uberis, reduces intracellular bacterial load, inhibits over-activation of the NF-κB pathway, and alleviates the inflammation and damage caused by S. uberis infection. This study increases our understanding of the mechanism through which taurine regulates autophagy and is the first to demonstrate the role of autophagy in S. uberis infected MAC-T cells. Our study also provides a theoretical basis for employing nutritional elements (taurine) to regulate innate immunity and control S. uberis infection. It also provides theoretical support for the development of prophylactic strategies for this important pathogen.

Interaction between HuR and circPABPN1 Modulates Autophagy in the Intestinal Epithelium by Altering ATG16L1 Translation

ABSTRACT Intestinal epithelial autophagy is crucial for host defense against invasive pathogens, and defects in this process occur frequently in patients with inflammatory bowel disease (IBD) and other mucosal disorders, but the exact mechanism that activates autophagy is poorly defined. Here, we investigated the role of RNA-binding protein HuR (human antigen R) in the posttranscriptional control of autophagy-related genes (ATGs) in the intestinal epithelium. We found that targeted deletion of HuR in intestinal epithelial cells (IECs) specifically decreased the levels of ATG16L1 in the intestinal mucosa. Intestinal mucosa from patients with IBD exhibited reduced levels of both HuR and ATG16L1. HuR directly interacted with Atg16l1 mRNA via its 3′ untranslated region and enhanced ATG16L1 translation, without affecting Atg16l1 mRNA stability. Circular RNA circPABPN1 blocked HuR binding to Atg16l1 mRNA and lowered ATG16L1 production. HuR silencing in cultured IECs also prevented rapamycin-induced autophagy, which was abolished by overexpressing ATG16L1. These findings indicate that HuR regulates autophagy by modulating ATG16L1 translation via interaction with circPABPN1 in the intestinal epithelium.

Emergence and populations genomics of the highly invasive chestnut blight pathogen

Invasive pathogens are a threat to forest and agroecosystems, as well as animal and human health. Identifying genomic determinants of pathogen evolution, as well as investigations into the genetic structure of invasive pathogen populations provide fundamental insights to why species can emerge as invasive pathogens. In this PhD project I investigated the emergence and population genomics of the invasive chestnut blight fungus Cryphonectria parasitica, using comparative and population genomic approaches. C. parasitica recently emerged as an invasive bark pathogen on non-Asian Castanea species in North America and Europe. In the first chapter, I investigated genomic determinants of lifestyle transitions in the genus Cryphonectria, by genome comparisons of C. parasitica and its sister species. The study uncovered a striking loss of genes associated with carbohydrate metabolism in the invasive pathogen C. parasitica, which may have promoted its pathogenicity on Castanea species. The second chapter explores the emergence and diversification of a highly invasive chestnut blight lineage across south-eastern Europe. By analyzing the genome-wide diversity of a large set of C. parasitica isolates of predominantly European origin, the study showed that a highly successful clonal pathogen lineage can emerge from a recombinant bridgehead population within Europe. Interestingly, the emergence of this clonal lineage was accompanied by an evolutionary transition from mixed mating type populations to single mating type outbreak populations. Lastly, in the third chapter I investigated temporal changes in genetic diversity of established C. parasitica populations in southern Switzerland, as well as potential links between the presence of the deleterious hyperparasitic mycovirus Cryphonectria hypovirus 1 (CHV1) and fungal genome-wide diversity. The results indicate increased mating among related fungal individuals, resulting in high genetic similarity of genotypes and facilitated CHV1 transmission. There were no substantial changes in fungal population structure and after ˜30 years and no detectable impact of CHV1 presence on fungal genome-wide diversity. Although our results show stable CHV1 incidence in fungal populations over three decades, the short-term interaction dynamics are likely highly volatile. The overall findings of this PhD thesis highlight the relevance of genomic determinants facilitating pathogen emergence and invasions. C. parasitica is a useful model to study fundamental questions of pathogen evolution and invasive processes, as well as antagonistic pathogen-hyperparasite interactions.