Date on Master's Thesis/Doctoral Dissertation

5-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Demuth, Donald

Committee Co-Chair (if applicable)

Uriarte, Silvia

Committee Member

Lawrenz, Matthew

Committee Member

Alard, Pascale

Committee Member

Graham, James E.

Author's Keywords

catecholamines; neutrophils; aggregatibacter actinomycetemcomitans; epinephrine; iron; Siglec-9

Abstract

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative facultative anaerobe and an opportunistic oral pathogen, strongly associated with localized periodontitis and other inflammatory diseases. Periodontitis is a chronic inflammation of the periodontium resulting from the inflammatory response of the host towards the dysbiotic microbial community present at the gingival crevice. The host immune response creates a hostile environment for microorganisms; therefore, it is important for Aa to be able to regulate the necessary genes to survive and thrive in such an environment. Aaexpresses several virulence factors such as a cytolethal distending toxin (Cdt), lipopolysaccharide (LPS) and leukotoxin A (LtxA), that allows it to evade the immune response. For example, LtxA, considered a major virulence factor, targets leukocytes by creating pores on their membranes leading to cell lysis. LtxA has also been shown to provide Aa resistance against internalization and killing by neutrophils, however the effect of the toxin on neutrophils at a non-lethal level is often overlooked. At the subgingival pocket Aa is in a tug-of-war scenario where immune cells will secrete molecules such as catecholamines (i.e., epinephrine) to sequester residual metals, so these are not available as nutrients. To compete for such mineral bacteria have evolved different mechanisms, such as two-component systems (TCS) that allow them to assess their environment and adjust accordingly. Aa expresses the QseBC TCS composed of the sensor protein QseC and the response regulator QseB. Previously, catecholamines and iron were identified as the signals that activate the QseBC TCS in Aa, necessary for the organism to acquire iron as a nutrient to survive in the anaerobic environment. However, the source of catecholamines had not been identified at the time. The main objectives presented in this dissertation are a) the characterization of epinephrine interaction with different components of QseBC TCS, b) to characterize the synthesis, storage, and release of catecholamines by neutrophils stimulated with Aa, and c) determination of the effect sublytic levels of LtxA has on neutrophils. Previously, our group showed that QseC, primarily the periplasmic domain of QseC, is required for biofilm formation and virulence. A third gene was found to be co-expressed in the qseBC operon, YgiW, a protein of unknown function, but essential for biofilm growth and virulence. Additionally, it was previously shown that in the presence of Cat-Fe the expression of the enterobactin operon remained unchanged. Among the genes in this operon there is the fepA gene encoding for the enterobactin receptor FepA. In this work we investigated the interaction of different components of the QseBC TCS and epinephrine. Using Aa mutants for the different QseBC component we determined that FepA, QseC and the QseC periplasmic domain are necessary for Aa to interact with epinephrine (Chapter 3). In contrast, YgiW was not required for epinephrine to interact with Aa. Due to the high infiltration of neutrophils that is characteristic of periodontitis we proposed neutrophils as the epinephrine source for Aa. We present evidence of the ability of human neutrophils to release epinephrine upon Aa challenge (Chapter 4). In addition, we demonstrated that epinephrine is stored in azurophilic granules and proposed that Aa gains access to it by inducing degranulation. To connect our findings, we demonstrated that chemically define media (CDM) supplemented with iron and epinephrine from human neutrophils promoted Aa growth and the induction of the qseBC operon. LtxA, a major virulence factor expressed by Aa, targets neutrophils and induces cell lysis. However, lysis only occurs when cells are exposed to high levels of the toxin, the sublytic effects of the toxin on neutrophils remains understudied. We show that the minimally leukotoxic Aa strain 652 caused minimal cytotoxicity in human neutrophils, even at MOI 50 (Chapter 5). Assessment of membrane permeability confirmed that human neutrophils suffered loss of membrane integrity. However, neutrophils were functionally active and were able to internalize Aa at MOI 10. In contrast, Aa internalization did not occur at MOI 50 for both the minimally leukotoxic strain or a LtxA mutant (JP2 DltxA). Aachallenge induced the formation of cytotoxic vacuoles, which have previously been associated with pore-forming toxins, but we observed cytoplasmic vacuolation regardless of LtxA expression of Aa strains. Siglec-9, a sialic acid-binding immunoglobulin-type lectin, has been associated with cytoplasmic vacuolation. Therefore, we measured Siglec-9 as an alternative inducer of cytoplasmic vacuolation. We were able to show that Aa 652 enhances Siglec-9 expression in human neutrophils and that IL-8 priming before Aa challenge further increases expression. The findings presented in this dissertation contribute to the current knowledge of nutrient acquisition mechanisms and provides the foundation for future studies of how Aa can modulates the inflammatory response of neutrophils through Siglec-9 activation.

Download

Share

COinS