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«Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of Nontypeable Haemophilus influenzae Colonization by Katherine Alice ...»

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Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of

Nontypeable Haemophilus influenzae Colonization


Katherine Alice Rempe

Department of Molecular Genetics and Microbiology

Duke University




Joseph W. St. Geme, III, Supervisor


Margarethe J. Kuehn


Patrick C. Seed


Raphael H. Valdivia Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University i v ABSTRACT Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of Nontypeable Haemophilus influenzae Colonization by Katherine Alice Rempe Department of Molecular Genetics and Microbiology Duke University Date:_______________________



Joseph W. St. Geme, III, Supervisor ___________________________

Margarethe J. Kuehn ___________________________

Patrick C. Seed ___________________________

Raphael H. Valdivia An


of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University Copyright by Katherine Alice Rempe Abstract Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculationof 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

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we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface.

We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked

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Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

Taken together we have shown that the HMW1 and HMW2 proteins are involved in colonization in vivo, that proper glycosylation and secretion of these proteins is needed for proper protein function, and that the unconventional N-linked glycosylation mechanism used by NTHi to modify HMW1 and HMW2 is also used by at least three other gram negative species to modify adhesive proteins. This work greatly enhances our understanding of NTHi adherence and colonization and demonstrates that findings regarding NTHi adherence are transferable to other species.

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List of Tables

List of Figures


1. Introduction

1.1 Organisms Used in these Studies

1.1.1 Nontypeable Haemophilus Influenzae

1.1.2 Kingella kingae

1.1.3 Aggregatibacter aphrophilus

1.2 Gram-Negative Bacterial Adherence

1.2.1 Pilus Adhesins Chaperone-Usher Pili Type IV pili Curli

1.2.2 Non-Pilus Adhesins Type V Secretion Type Va (Classical) Autotransporters Type Vb Two Partner Secretion Systems Type Vc (Trimeric) Autotransporters

1.3 Nontypeable Haemophilus influenzae Adherence

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1.4 Kingella kingae Adherence

1.5 Aggregatibacter aphrophilus Adherence

1.6 Bacterial Protein Glycosylation

1.6.1 O-Linked Glycosylation

1.6.2 N-linked Glycosylation

1.6.3 HMWC-mediated N-linked Glycosylation

1.7 Thesis Overview

2. The HMW1 and HMW2 adhesins enhance the ability of nontypeable Haemophilus influenzae to colonize the upper respiratory tract of rhesus macaques

2.1 Introduction

2.2 Methods

2.2.1 Bacterial Strains and Culture

2.2.2 Adherence Assays

2.2.3 Animal Studies

2.2.4 Bacterial Recovery

2.2.5 ELISA

2.2.6 SDS-PAGE

2.2.7 Statistics

2.3 Results

2.3.1 NTHi adheres to rhesus macaque respiratory epithelial cells in vitro............... 37 2.3.2 NTHi is able to colonize the upper respiratory tract of rhesus macaques........ 38

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2.3.4 Colonization is an immunizing event

2.4 Discussion

3. Determinants of HMW1 Function

3.1 Introduction

3.2 Methods

3.2.1 Bacterial Strains and Growth Conditions

3.2.2 Purification of untagged HMW1

3.2.3 Purification of YebF fused HMW1

3.2.4 Western Blots

3.2.5 Native Gel Electrophoresis

3.2.6 Circular Dichroism

3.3 Results

3.3.1 The Pattern of Glycosylation on HMW1 is Critical for Protein Function.......... 61 3.3.2 HMW1 purified from the bacterial cell surface is functional while HMW1 purified from the cytoplasm is non-functional.

3.3.3 Secretion alone is not sufficient to produce functional HMW1

3.4 Discussion

4. Unconventional N-linked glycosylation promotes trimeric autotransporter function in Kingella kingae and Aggregatibacter aphrophilus.

4.1 Introduction

4.2 Methods

4.2.1 Strains and culture conditions

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4.2.3 Homologue identification:

4.2.4 Adherence assays

4.2.5 Autoaggregation assays

4.2.6 Statistical analysis

4.2.7 Isolation of outer membrane proteins

4.2.8 Formic acid treatment of outer membrane proteins

4.2.9 SDS PAGE

4.2.10 In-gel digestion

4.2.11 Mass spectrometry analysis

4.2.12 Database searching

4.2.13 Immunogold labeling and Transmission electron microscopy

4.3 Results

4.3.1 Kingella kingae and Aggregatibacter aphrophilus have HMW1C homologues

4.3.2 HMW1CKk is required for K. kingae adherence to human epithelial cells and autoaggregation

4.3.3 HMW1CKk glycosylates the trimeric autotransporter Knh

4.3.4 Co-expression of Knh and HMW1CKk in E. coli is sufficient to facilitate bacterial adherence to epithelial cells

4.3.5 Mutating hmw1CKk decreases the amount of Knh on the bacterial cell surface

4.3.6 HMW1CAa is necessary for A. aphrophilus autoaggregation and adherence to human epithelial cells

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4.3.8 EmaA facilitates autoaggregation and bacterial adherence to host cells.......... 99

4.4 Discussion

4.5 GenBank Accession Numbers

5. Future Directions

5.1 Colonization of rhesus macaques with additional strains of NTHi

5.2 Protection by colonization-induced antibody

5.3 Structural analysis of HMW1

5.4 Investigation of additional key modified residues of HMW1

5.5 Specificity of HMW1C for HMW1

5.6 Small molecule inhibitor of HMW1C



Peer Reviewed Publications

Book Chapters

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Table 2: Animal Weights over the Course of the Experiment

Table 3: Bacterial Strains Used in Chapter 3

Table 4: Bacterial Strains used in Chapter 4

Table 5: Glycopeptides identified with di-hexose modification in Knh

Table 6: Glycopeptides identified with di-hexose modification in Knh

Table 7: Glycopeptides identified in EmaA

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Figure 2: Type IV Pilus Biogenesis

Figure 3: Curli Biogenesis

Figure 4: Type Va Classical Autotransporter

Figure 5: Type Vb Two Partner Secretion System

Figure 6: Type Vc Trimeric Autotransporter

Figure 7: Model of K. kingae adherence

Figure 8: Comparison of Conventional and HMW1C-Mediated N-linked Glycosylation

Figure 9: NTHi adherence to rhesus macaque bronchial epithelial cells is dependent on the HMW1 and HMW2 proteins.

Figure 10: An inoculum of 105 CFU of NTHi strain 12 results in consistent colonization of rhesus macaques.

Figure 11: HMW1 and HMW2 in NTHi colonization of rhesus macaques

Figure 12: HMW1 and HMW2 independently facilitate colonization of rhesus macaques

Figure 13: Colonization with NTHi can be an immunizing event

Figure 14: Pattern of glycosylation affects HMW1 function.

Figure 15: There are at least two specific sites that must be modified for HMW1 to function

Figure 16: Glycosylation pattern impacts protein structure.

Figure 17: HMW1 obtained from the bacterial surface is structurally different than HMW1 obtained from the cytoplasm.

xiii Figure 18: Non-HMW1B depended secretion of HMW1 results in non-functional protein.

Figure 19: HMW1CKk and HMW1CAa are highly homologous to HMW1C

Figure 20: Expression of HMW1CKk is required for adherence and autoaggregation...... 90 Figure 21: HMW1CKk is required for Knh expression on the surface of K. kingae............. 95 Figure 22: HMW1CAa expression is required for A. aphrophilus adherence and autoaggregation

Figure 23: EmaA is an adhesin and involved in autoaggregation

Figure 24: Plasmid Expression of HMW1C complements a HMW1C Genomic Deletion

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succeed in this endeavor most especially my lab-mates Eric Porsch, Jessica McCann, and Susan Grass. I’d also like to acknowledge all the other scientists I worked with over the past five years at Duke and at CHOP who have been willing to share their knowledge with me, especially Dr. Steve Seeholzer, Dr. Jolaine Wilson, and Lynn Spruce. I am grateful to Dr. Philip Johnson and Dr. James Wilson who allowed me to use their nonhuman primates for the experiments described in Chapter 2. I’d like to thank my family and friends for encouraging me along the way. Finally, I’d like to thank my mentor, Dr.

Joesph W. St. Geme III, for challenging me to be a great scientist, teaching me the skills I needed, and allowing me to investigate my own ideas.

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Haemophilus influenzae was first isolated by Richard Pfieffer during the 1889 influenza pandemic and was believed to be the microorganism responsible for influenza (Pfeiffer 1892). In 1933 it was determined that influenza was of viral etiology rather than bacterial; however, H. influenzae retained its name in homage to the circumstance of its discovery. In 1917, the species was given its current name, Haemophilus influenzae, with the genus Haemophilus recognizing its affinity for blood.

H. influenzae is a member of Pasturellaceae and the type-species for the genus Haemophilus and is a non-spore forming, non-motile gram-negative coccobacillus. It is able to grow both aerobically and anaerobically and requires hemin (factor X) and nicotinamide adenine dinucleotide (NAD) (factor V) under aerobic conditions. H.

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