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Applying phenotypic phage selection to Chlamydia trachomatis infection


Type

Thesis

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Authors

Adams, Simone 

Abstract

Chlamydia trachomatis (Ctr) is an obligate intracellular pathogen that causes the most common bacterial sexually transmitted infection worldwide and is the agent of trachoma, an ocular infection that is the leading cause of preventable blindness. Ctr has a biphasic lifecycle dependent on an infectious extracellular form, the elementary body (EB), and a non-infectious replicative form, the reticulate body (RB). EBs induce entry into host cells and reside within a specialised vacuole termed an inclusion where EBs differentiate into RBs. Following replication and redifferentiation from RB to EB, Ctr bacteria exit the host cell by stimulating host cell lysis or extrusion of the inclusion. Ctr virulence proteins are translocated into the host cell by EBs and RBs, and subvert host cell functions from the inclusion membrane, host cytosol or the inclusion lumen to propagate this cycle.

The detection of Ctr infection remains challenging, and no vaccine is available. As the obligate intracellular lifecycle of Ctr is more reminiscent of viral than most other bacterial infections, bacterial entry and exit might present opportunities for intervention. Monoclonal antibodies (mAb) have recently been identified that enable the detection and treatment of viral infectious diseases but have yet to be comprehensively applied to study bacterial infections. Such mAbs can be identified by target based or phenotypic phage display.

Here, phenotypic phage display was performed in two ways: (1) on a population of cells infected with Ctr or (2) on EB-enriched material derived from lysed, infected cells. In (1), panning for antigens was performed on the surface of infected cells to understand how antigen exposure at the plasma membrane may be altered by intracellular infection. Antibodies identified by this screen provided insights into the challenges of selection and revealed two differentially expressed surface antigens that signify Ctr infection.

In selection (2), panning was performed on material released from infected cells including intact, asynchronous bacteria, and host and chlamydial antigens. Antibodies derived from this screen specifically recognised Ctr-infected cells when probed by immunofluorescence. Three distinct binding patterns (diffuse, inclusion-recognising, or inclusion-recognising with filaments) were evident. By immunofluorescence, four of the antibodies recognized a punctate structure within the inclusion lumen at 24 – 48 hours post infection (hpi) which then expanded to form a novel vesicular structure, primarily by 56 hpi. The vesicles are constant in diameter and most abundant in inclusions nearly devoid of bacteria. These previously unrecognised sparsely populated inclusions appear to develop during infection as while 7% of inclusions are sparse at 24 hpi this increases to 30% at 56 hpi. Characterization of the vesicles by microscopy indicates that they are membrane-enveloped structures with distinct composition to the inclusion membrane that sometimes colocalize with canonical bacterial membrane markers. Co-immunoprecipitation and mass spectrometry were used to identify the protein target of one antibody. Interestingly, binding to a phospholipid array revealed the antibody also strongly recognized phosphatidylinositol (5) phosphate.

This study has demonstrated that the application of phage display to Ctr infection can be a valuable tool to study intracellular infections. The antibodies derived from the screen have provided insights to the Ctr lifecycle and host-pathogen interactions. The identification of a novel structure within the inclusion reveals how little is known during the late stage of the lifecycle, and the findings raise questions about the fate of the inclusion after infection when lysis does not occur. In the future, this technology could be applied to other intracellular pathogens to better understand their functions and to potentially create therapeutics.

Description

Date

2023-07-31

Advisors

Hayward, Richard

Keywords

antibody, Chlamydia trachomatis, infection, phage display, phage selection

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
AstraZeneca