Manipulation of host nuclear function by Chlamydia trachomatis

Martin, Oliver W. F. (2016) Manipulation of host nuclear function by Chlamydia trachomatis. Doctoral thesis, Birkbeck, University of London.

Abstract

Chlamydia trachomatis is an obligate intracellular bacterium, responsible for the most prevalent bacterial sexually transmitted infection and is the leading cause of acquired blindness worldwide. Chlamydiae survive and replicate within a specialised intracellular membrane-bound compartment known as the inclusion. Inclusion biogenesis requires manipulation of host processes by translocated bacterial effectors. Despite Chlamydia targeting at least one effector into the nucleus, surprisingly little is known about how and why the bacteria interact with this major organelle. The aim of the work presented in this thesis was to examine the consequences of Chlamydia infection on nuclear architecture, organisation and function. Structurally, nuclei of cells infected with C. trachomatis become highly lobulated, reminiscent of both laminopathy and transformed cells. Analysis of the nuclear lamina (NL) demonstrated that lamin A/C is enriched at the inclusion-proximal face of the nuclear envelope (NE) and depleted elsewhere around the nuclear periphery. Lamin B is reduced and nuclear pore complexes (NPCs) are depleted in the inclusion-proximal lamin A/C-enriched region. Other NL scaffolding proteins including SUN proteins and emerin are unaffected. Transmission electron microscopy revealed a minimal distance of ~100 nm is maintained between the NE and inclusion membrane (IM), showing a classical membrane contact site does not form. Lamin A/C enrichment and NPC depletion occur independently of existing cellular NPC-free islands present in the NE, and are specific to C. trachomatis rather than C. muridarum. Functionally, these infection-associated NE alterations are promoted by bacterial factors and can recover following induced bacterial killing or inclusion clearance. Host nuclear protein transport ceases in the NPC-depleted region, whereas host nuclear mRNA distribution is unaffected. Associated local changes in host chromatin architecture occur, since heterochromatin accumulates at the inclusion-proximal face of the nuclear periphery at NPC-depleted and lamin A/C-enriched zones. Lamin A/C changes are the driving force behind the NE modifications, as the NPC-depletion and heterochromatin accumulation is absent when cells lacking lamin A/C are infected with C. trachomatis. Remarkably, in these knockout cells, inclusions are larger and more bacterial progeny are produced. Computationally, chlamydial proteins of unknown function that may be translocated to the nucleus or NE were identified. A tool was developed to generate a consensus secondary sequence from multiple available prediction software. Using this tool, the chlamydial proteome was searched for proteins similar in secondary structure to known cellular NE or NE-interacting proteins. Three proteins were identified, of which CT350 and CT384 are predicted to be type III secretion substrates. When ectopically expressed in HeLa cells, CT384 localised to the nucleus and resulted in aberrant nuclear architecture. Taken together, the data demonstrate that Chlamydia profoundly manipulates nuclear architecture from within the inclusion, and via interactions at the inclusion-proximal region of the NE constructs a lamin A/C-enriched platform to drive localised redistribution of heterochromatin and changes in host gene expression.

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