Neutrophils are among the first white blood cells recruited to the site of infection once microbial pathogens enter the host organism. At site, they perform a well-orchestrated chain of processes that aims to kill the microbial invader. Most prominent, neutrophils engulf microbes to inactivate them intracellularly, a process called phagocytosis. Alternatively, neutrophils can release neutrophil extracellular traps (NETs). NETs consist of chromatin decorated with antimicrobial effector proteins – a structure that can entangle bacteria and fungi. Neutrophils are crucial during fungal infections. This is reflected in the increased risk of fungal infections resulting of neutropenia. The concept of nutritional immunity describes every infection as a battle for resources. Those are mostly metal trace elements. For a long time, neutrophils were seen as powerful, but “mindless”, killers with a limited set of actions and no transcriptional capacity, but this view is in the flux. In the presented thesis, it was my goal to gain new insights into the interplay of neutrophils and fungi – with special attention to metal-nutritional aspects. We compared human neutrophils lacking the ability to undergo NETosis, due to a non-functional NADPH complex, and neutrophils from the same person that were “cured” by gene therapy. We investigated those NETs and found that their inhibitory activity towards the mold A. nidulans depends on calprotectin, a known zinc-chelator. Considering the high influx of neutrophils, we wanted to unravel the neutrophils’ contribution to the metal milieu at the site of infection and trace element changes resulting from NETosis. By combining synchrotron radiation XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial element distribution in activated neutrophils and NETs. Most strikingly, we found neutrophils to be exceptionally high in Fe and the process of NETosis to be reducing available Zn in the surrounding and the early phagosome, possibly by the formation of Zn-rich vesicles. Using RNA-sequencing, we analyzed the interplay of the C. albicans and neutrophils face-to-face. We dissected their transcriptional profile and revealed a manifold response in neutrophils that include cytokine induction and cellular rearrangement. We further were the firsts to explore the transcriptional response of C. albicans to NETs. Our data indicates a distinct response compared to intact neutrophils or other known stress triggers. Metal homeostasis was affected in Candida in both set-ups. In summary, this thesis provides new insights into the interaction of fungal pathogens with neutrophils and emphasizes the impact of nutritional aspects on this interplay. A deeper understanding of the nutritional immunity during fungal infection might open up new strategies to tackle fungal infections – a growing threat worldwide.

No description available.

Crohn’s disease (CD) is an inflammatory disease of the gastrointestinal tract attributed to an aberrant immune response to environmental and microbial triggers. Individuals with CD exhibit an enrichment of pro-inflammatory strains of Adherent-Invasive E. coli (AIEC) and often report a relapse of symptoms following a period of acute psychological stress. Despite a known immunosuppressive role, the mechanism by which stress contributes toward the development and progression of intestinal inflammation remains unknown. Here, we use a well characterized model of restraint stress to investigate the influence of psychological stress on host protection against a CD-associated strain of AIEC. We found that stress results in profound intestinal dysbiosis, allowing for a complete dominance of Enterobacteriaceae. Interestingly, while stress alone drives a state of low-grade inflammation and loss of barrier integrity in the gut, in the presence of a pathobiont strain of AIEC, stress drives a substantially heightened inflammatory response which exacerbated the resultant loss of barrier integrity. Moreover, we have found stress induces an augmented nutritional immune response, providing AIEC a competitive niche against commensal bacteria lacking alternative methods of iron uptake. Further, we see that stress-induced glucocorticoids mediate broad apoptosis of the CD45+CD90+ lymphocytic population in the gut. The loss of this population prevents an appropriate IL-22 mediated response to dysbiosis. Accordingly, blocking glucocorticoid signalling or exogenous administration of IL-22 prevents the stress-induced expansion of AIEC. This work underscores the complex nature of psychological stress such that the combination of iron limitation and glucocorticoid mediated immune attrition are simultaneously required for the stress-induced expansion of AIEC. These findings present novel insight into the mechanistic consequences of glucocorticoid signalling on impaired immune function and the provision of an inflammatory environment, resulting in a distinct impact on CD susceptibility. As such, deeper insight regarding the complex underpinnings of CD will assist in efforts to design representative models and will strengthen the discovery of targeted therapeutics. / Thesis / Master of Science (MSc) / Crohn’s disease (CD) is an inflammatory disease of the gastrointestinal tract resulting from an exaggerated immune response. CD patients often report a relapse of symptoms following a period of psychological stress and are at an increased likelihood of having pro-inflammatory strains of E. coli within their gut. Here, we use a model of restraint stress to investigate how psychological stress modulates the abundance of bacterial species associated with CD. We found stress results in the limitation of essential nutrients, allowing for an outgrowth of E. coli. Further, stress hormones lead to the loss of a protective immune response in which E. coli expansion can be prevented by blocking these hormones or restoring immune signalling. Together, we conclude that stress leads to immune cell death and creates an iron limited environment that favours E. coli expansion. Such work begins to uncover the functional consequence of stress and its’ role in disease progression.

During the 20th century, significant strides were made in curtailing the burden of childhood diarrhea, including advances in vaccine research, the advent of antibiotics, improved water and sanitation, and expanded access to health information across the globe. Despite this progress, today diarrhea ranks second only to pneumonia as a leading cause of mortality in children under five years, with a disproportionate burden of 90% of diarrheal deaths in South Asia and Sub-Saharan Africa. Additionally, substantial morbidity due to diarrhea persists in young children, with more than 45 million disability-adjusted life years (DALYs) lost due to diarrhea in 2015. Long-term consequences of childhood diarrhea include undernutrition, impaired gut function, altered gut microbiota, and compromised cognitive development. The 21st century presents an opportunity to eliminate the health disparity affecting millions of children suffering disproportionately from preventable diarrheal diseases. Recent advances in molecular laboratory technology have enabled detailed assessment of diarrheal burden and etiology, illuminating the highest burden pathogens for focused interventions. Among the top diarrheal pathogens, rotavirus (RV) is the leading cause of diarrhea-attributable death in the first year of life. While we have vaccines against RV, these vaccines consistently underperform in low and middle-income countries (LMICs) with efficacy of 18% to 61% compared to > 85% efficacy in high income countries. Reasons for rotavirus vaccine underperformance remain unclear, and no vaccines are available for other high burden diarrheal pathogens. This requires consideration of complementary and alternative interventions for diarrhea prevention. To assess factors related to rotavirus vaccine performance, we enrolled a 700-infant birth cohort in an urban slum of Dhaka, Bangladesh, in the Performance of Rotavirus and Oral Polio Vaccines in Developing Countries (PROVIDE) study: a randomized controlled trial of a 2-dose monovalent oral rotavirus vaccine (RV1). With a primary outcome of any rotavirus diarrhea (RVD) post-vaccination to one year, we conducted biweekly home-based diarrhea surveillance with rotavirus antigen detection in diarrheal stools by ELISA. We found RV1 efficacy of 51% (95% CI 33.8–63.7) in per protocol analysis. Importantly, among 12 explanatory variables tested for association with RVD, serum zinc concentration (SZC) in infants at week 18 associated with risk of RVD up to one year (OR 0.77, 95% CI 0.66–0.91), independent of vaccination status. This finding led to broader investigation of the relationship between zinc status and diarrhea in the PROVIDE cohort. Among 577 PROVIDE infants, 16.5% were zinc deficient at week 18 (SZC < 65μg/dL). By logistic regression, zinc deficient infants had increased odds of diarrhea in the first year of life compared to zinc replete infants (OR 2.76, 95% CI 1.08–7.04), and they were nearly 4 times more likely to have diarrhea of viral etiology (OR 3.94, 95% CI 1.55–10.03). Furthermore, in Kaplan Meier analysis we found a strong correlation between zinc deficiency and time to first episode of viral diarrhea (median survival 27 vs 33 weeks in zinc deficient vs non-deficient infants, p Our results indicate further consideration of zinc as a critical and modifiable co-factor in ameliorating the burden of childhood viral diarrhea. Carefully designed trials of zinc supplementation interventions could determine whether zinc may fill the gap in protection against childhood viral diarrhea, and inquiries into the zinc-diarrhea molecular pathway could elucidate mechanisms for focused development of future interventions.

Mastitis resulting from mammary gland infection is a common and painful disease associated with lactation. In addition to the impact on human and animal health, mastitis causes substantial economic losses in the dairy industry. Staphylococcus aureus is a frequent cause of mastitis worldwide. Despite significant progress in understanding S. aureus pathogenesis in general, much remains to be learned regarding virulence factors relevant in the context of mastitis. In mammary gland infections, it is not fully understood which metal acquisition systems are required for S. aureus survival. To help understand molecular mechanisms by which S. aureus might acquire essential metals, such as iron, within lactating mammary glands, S. aureus mutants were tested for growth defects in vitro. A low-iron media (TMM) was created and supplemented with differing iron sources relevant to mastitis infection such as host iron-binding proteins lactoferrin and transferrin. Mutants were grown in the various iron sources to determine which genes were involved with iron acquisition for each specific media tested. Results show that a double knock-out (∆htsA/sirA::ba) involved with two iron siderophore receptors and the ATPase, fhuC::ba, which powers those receptors are essential for growth in media supplemented with human lactoferrin, while mutants involved with the iron siderophore Staphyloferrin B (sbnE::ba) and its specific receptor (sirA::ba) proved important for growth in bovine lactoferrin. Additionally, S. aureus mutants were grown in bovine and human milk. Significant growth defects in human milk were found for mutants involved with zinc (znuBC::ba) and manganese (psaA::ba) acquisition. Iron limitations leading to growth defects were also found in ∆htsA/sirA::ba and fhuC::ba grown in human milk. Growth defects in bovine milk were seen for psaA::ba but not zinc genes. Growth of the fhuC mutant was shown to be significant, but not the double knock-out, indicating that iron acquisition in bovine milk does not involve the SirABC or HtsABC siderophore receptors. A mutant involved in purine synthesis, purH::ba, was also shown to have a significant growth defect in bovine milk. The importance of S. aureus metal acquisition has been well established, but there is a significant need to research these multifaceted processes further. Increased understanding of how metal acquisition facilitates bacterial survival in the lactating mammary gland can provide therapeutic targets for more effective mastitis prevention and treatment.

No description available.

Neisseria gonorrhoeae is an obligate human pathogen that causes the common STI gonorrhea, which presents a growing threat to global health. The WHO estimated 78 million new cases of gonorrhea worldwide in 2017, with estimates of 820,000 new cases in the United States alone according to the CDC. High-frequency phase and antigenic variation inherent in N. gonorrhoeae, coupled with its natural ability to rapidly acquire and stably integrate antimicrobial resistance factors into its genome, have culminated in an infection against which there is no effective vaccine, and for which the list of viable therapeutic options is quickly shrinking. Moreover, no protective immunity against subsequent infections is elicited upon exposure to N. gonorrhoeae, which highlights the need for research of novel antimicrobial and vaccination strategies. Within the human host, N. gonorrhoeae utilizes a unique strategy to overcome host sequestration of essential nutrients – termed nutritional immunity (NI) – such as ions of trace metals. The pathogen produces a family of outer membrane proteins called TonB-dependent transporters (TdTs) capable of binding to host NI factors and stripping them of their nutritional cargo for use by the pathogen. Importantly, these TdTs are very highly conserved and expressed among Neisseria species. TbpA is a well-characterized TdT that allows N. gonorrhoeae to acquire iron from human transferrin, and recent studies from our lab have shown that TdfH is capable of binding to a zinc-sequestering S100 protein called calprotectin and stripping it of its zinc ion. The S100 proteins are EF-hand calcium-binding proteins that naturally play an integral role in Ca2+ homeostasis, but due to their ability to bind transition metals, they have also demonstrated an innate immunity role by participating in nutrient sequestration. The S100 proteins are expressed in all human cells, and all are capable of binding transition metals including zinc, manganese, and cobalt. Calprotectin, S100A7, and S100A12 have demonstrated an ability to hinder the infection potential of pathogenic E. coli, S. aureus, C. albicans, and various other pathogens via zinc sequestration. Herein, we demonstrate that N. gonorrhoeae is able to overcome this phenomenon and actually utilize these proteins as a zinc source in vitro. Furthermore, we identify S100A7 as the specific ligand for TdfJ, which utilizes this ligand to internalize zinc during infection. S100A7 growth support in vitro is dependent upon a functional TonB, TdfJ, and the cognate ABC transport system ZnuABC, and isogenic mutants incapable of producing znuA or tdfJ recover S100A7 utilization by complementation. Whole-cell binding assays and affinity pulldowns show that S100A7 binds specifically to both gonococcal and recombinant TdfJ, and growth and binding experiments show that these described phenomena are specific to human and not mouse S100A7. Finally, we show that a His-Asn double mutant S100A7 that is incapable of binding zinc cannot be utilized for growth by gonococci. These data illustrate the unique nature of the gonococcus’ ability to co-opt host defense strategies for its own purposes, and further identify the TdTs as promising targets for strategies to combat and prevent gonococcal infection.