Functional Characterization of the Epithelial Protein, PRAP1, and Generation of Human Microbiota-Associated Mice to Model Bacterial Vaginosis Open Access

Wolfarth, Alexandra (Summer 2020)

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The microbiota maintains critical interactions with the host at the mucosa of both the gastrointestinal tract and the female reproductive tract. To understand the mechanisms by which certain members of the microbiota promote intestinal and reproductive health, we developed two independent projects. We first functionally characterize an under-studied protein, Proline-rich acidic protein 1 (PRAP1), induced by Lactobacilli in the gut epithelium. We found PRAP1 is a 17 kDa intrinsically disordered protein that prolongs the survival of mice after total body irradiation and prevents irradiation-induced apoptosis in the gut epithelium. We conclude that PRAP1 is an intrinsically disordered protein highly expressed by the gastrointestinal epithelium and functions at exposed surfaces to protect the barrier from oxidative insult. Second, to further understand the mechanisms by which the vaginal microbiota promote female reproductive health, we sought to improve current mouse models of the vaginal microbiota. We generated human microbiota-associated (HMA) mice using vaginal swabs collected from pregnant women with or without bacterial vaginosis (BV). Our goal was to generate mice that had a vaginal microbiota and birth outcome comparable to their respective human donors. There was considerable variability in the microbes that colonized each mouse, with no association to the microbiota of the donor. Although human mothers with BV had more frequent adverse birth outcomes, the vaginal microbiota was not predictive of adverse birth outcomes in mice. Together, these projects further our understanding of the mechanisms by which the microbiota promotes intestinal health and the potential uses and limitations of mouse models involving the female reproductive tract.

Table of Contents

Chapter 1

The Small Intestinal Epithelium: A Dynamic Barrier Critical to Human Health---------------- 1

Introduction-------------------------------------------------------------------------------------- 1

Microbial Composition and Epithelial Cell Types of the Small Intestine------------------------ 2

Maintenance of the Epithelial Barrier------------------------------------------------------------ 4

Innate Immunity at the Small Intestinal Epithelium--------------------------------------------- 6

Mucus Layer----------------------------------------------------------------------------------- 6

Epithelial sensing of Microbes----------------------------------------------------------------- 7

Antimicrobial proteins------------------------------------------------------------------------ 12

Table 1. Major Antimicrobial Proteins Found in the Intestine----------------------------- 14

The Crypt-Stem Cell Niche--------------------------------------------------------------------- 15

Figure 1. Paneth cell function is critical for gut homeostasis------------------------------ 19

Inflammatory Bowel Disease------------------------------------------------------------------- 21

Chapter 2

Proline-rich acidic protein1 (PRAP1) protects the gastrointestinal epithelium from irradiation-induced apoptosis-------------------------------------------------------------------- 25

Abstract----------------------------------------------------------------------------------------- 26

Introduction------------------------------------------------------------------------------------- 27

Results------------------------------------------------------------------------------------------ 29

Figure 1. PRAP1 is an intrinsically disordered protein conserved in placental mammals- 30

Figure 2. PRAP1 is highly expressed by the epithelium of the gastrointestinal tract in mice and humans    32

Figure 3. Prap1-/- mice have an altered microbiota in the small intestine------------------ 36

Figure 4. Prap1-/- mice have elevated inflammation but no significant intestinal barrier defect        38

Figure 5. Prap1-/- mice are more susceptible to radiological challenge and have increased apoptosis in the intestinal epithelium--------------------------------------------- 41

Figure 6. PRAP1 protects enteroids from irradiation-induced apoptosis by limiting p21 expression 44

Discussion--------------------------------------------------------------------------------------- 45

Methods----------------------------------------------------------------------------------------- 50

Supplemetal Figures---------------------------------------------------------------------------- 59

Supplemental Figure 1. Generation and validation of PRAP1 recombinant protein, PRAP1 antisera and Prap1-/- mice--------------------------------------------------- 59

Supplementary Figure 2. PRAP1 is highly expressed in the murine and human endometrium         61

Chapter 3

A human microbiota-associated murine model for assessing the impact of the vaginal microbiota on pregnancy outcomes---------------------------------------------- 62

Abstract----------------------------------------------------------------------------------------- 63

Introduction------------------------------------------------------------------------------------- 64

Results------------------------------------------------------------------------------------------ 67

Table 1. The clinical parameters of the 19 pregnant women used for HMA mouse generation         68

Figure 1. Pregnant women with bacterial vaginosis harbor a distinct microbiota community structure        70

Figure 2. Generation of human microbiota-associated (HMA) mice harboring the microbiota collected from the vaginal tract of pregnant women with bacterial vaginosis-------------- 73

Figure 3. Pregnancy outcomes in human microbiota-associated (HMA) mice harboring the microbiota collected from the vaginal tract of pregnant women with bacterial vaginosis---- 75

Figure 4. Pregnancy outcome in human microbiota-associated (HMA) mice is associated with altered uterine cytokine levels during pregnancy----------------------------------------- 78

Discussion--------------------------------------------------------------------------------------- 79

Methods----------------------------------------------------------------------------------------- 83

Chapter 4

Discussion---------------------------------------------------------------------------------------------------------- 88

The Role of PRAP1 in the Epithelial Response to Irradiation--------------------------------- 88

PRAP1 Function in the Uterus----------------------------------------------------------------- 93

Lactobacilli are Beneficial in both the Gut and Reproductive Tract--------------------------- 94

Limitations of using Animal Models to Study the Human Vaginal Microbiota---------------- 96

References-------------------------------------------------------------------------------------------------------- 102

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