Shigella flexneri and Autophagy

Figure 1.6: Helical wheel scheme of a parallel dimeric coiled coil Hydrophobic interactions between residues a and d stabilize the core.

Ionic interactions between residues e and g give specificity to the coiled coil.

Golgi-resident Arl1. How can such a small protein exhibit so many differ-ent functions? SCOC contains only one functional domain, thus, the answer to the diversity of interactions and functions must rely in the nature of SCOC coiled coil. Hence, it was my aim to determine the SCOC ccd structure by X-ray crystallography. This included the expression and purification of SCOC constructs suitable for crystallography. These constructs were further charac-terized by biophysical and biochemical methods, providing insights into the stability and oligomerization state of SCOC. In addition, the interactions of SCOC with FEZ1 and Arl1 were analyzed with different methods.

1.3 Shigella flexneri and Autophagy

1.3.1 Selective Autophagy: Xenophagy

Autophagy is of great medical interest since it is not only a major target of cancer research, but also plays an important role in the innate and adaptive immunity response of higher eukaryotes (reviewed in Levineet al.[60], Deretic

& Levine [61]). Xenophagy is, like other selective autophagy pathways, me-diated through autophagy adaptors. Adaptors function by binding both the autophagic cargo and to a LC3 protein family member by a LC3-interacting

12 Introduction region (LIR). Sequestosome 1 (SQSTM1/p62) [62], nuclear dot protein 52 kDa (NDP52) [63–65], optineurin (OPTN) [66, 67], and neighbor of BRCA1 gene 1 (NBR1) [68] are adaptor proteins involved in the autophagic clearance of pathogens [69, 70]. All of these adaptors contain a ubiquitin binding domain, by which they recognize their ubiquitinated substrate [70].

Xenophagy plays a role in intracellular infections with various bacteria, e.g.

Shigella, Mycobacteria, Salmonella, Listeria, and Legionella [69]. The escape mechanisms of pathogens as well as the host cell’s defensive mechanism are di-verse. For example, Mycobacterium tuberculosis survives in phagosomal com-partments after invading the host cell by arresting fusion of the phagosome with the lysosome [71, 72]. Virulent Mycobacteria strains can also resist and inhibit autophagy, but this inhibition can be overcome by the induction of autophagy through various stimuli [73, 74].

In Salmonella infection, the bacterium survives in a Salmonella-containing vacuole, in whichS. typhimurium can replicate [75]. It secrets several effectors through its type III secretion system (TTSS), which results in bacteria invading the cytosol. S. typhimurium in the cytosol are rapidly polyubiquitinated and then recognized by the respective cargo adaptor NDP52 [63, 69].

1.3.2 Escape of intracellular Shigella flexneri from autophagy

Shigella flexneri is a human pathogen causing bacillary dysentery Shigellosis.

This mucosal bacterium has versatile instruments that circumvent the host cell immune response (reviewed in Ashida et al. [76]). It disrupts the initial vacuolar membrane surrounding the bacterium, it multiplies in epithelial cells, invading cells by exploiting actin polymerization. It manipulates the host cell death and signalling pathways. It has adapted a sophisticated mechanism to escape autophagy (see Figure 1.7). Shigella’s outer membrane protein VirG, which is crucial for its actin-based motility [77, 78], has a binding site for the host protein Atg5, by which autophagic destruction of the pathogen is triggered. However, Shigella secrets IcsB through its TT3S, which masks the binding site of Atg5 on VirG [78]. Mutant Shigella bacteria lacking IcsB were enwrapped by multilamellar structures positive for LC3 more frequently, as observed by EM. In vitro interactions of VirG-IcsB and VirG-Atg5 were confirmed by pulldown assays. Both VirG and IcsB originate from Shigella flexneri’s large virulence plasmid. VirG triggers autophagy, whereas IcsB did not reduce overall autophagy levels [78].

1.3 Shigella flexneri and Autophagy 13

Figure 1.7: Mechanism of Shigella flexneri’s escape from xenophagy

VirG on the outer membrane ofShigella contains a common binding site for Shigella’s virulence effector IcsB and the host cell’s protein Atg5.

IcsB masks the binding site, protecting Shigella from degradation by au-tophagy. InΔIcsB strains, VirG is recognized by the autophagic machin-ery through interaction with Atg5 and entrapped by autophagosomes.

In addition to this escape mechanism, Shigella also induces autophagy via Shiga toxins, resulting in cell death in an autophagy-dependent manner [79].

Remnants of the disrupted vacuolar membrane are targeted to autophagy via ubiquitination and interaction with p62 and LC3 [80]. Recently, also the cy-toskeleton has been involved in the host cell’s response to Shigella. Cytosolic Shigella are trapped in septin cages [81] andShigella are targeted to an actin and septin-dependent autophagic pathway, which requires p62 and NDP52 [64].

1.3.3 The autotransporter protein VirG

VirG, also connoted as IcsA, is an autotransporter protein with the typical domain structure of a type Va autotransporter. It features a N-terminal sig-nal sequence (1–52), a passenger domain (53–758) and a transmembrane porin domain (759–1102). The secretion mechanism of type Va autotransporters is depicted in Figure 1.8. In the bacterial cytosole, VirG is stabilized by chaper-ones, e.g. DnaK [83]. VirG is translocated through the inner bacterial mem-brane by the Sec machinery. The signal sequence is cleaved in the periplasm,

14 Introduction

Figure 1.8: Secretion mechanism of type Va autotransporters Figure was modified from Junker et al. [82]. Copyright (2006) National Academy of Sciences, USA

where VirG is chaperoned by Skp [84]. The transmembrane porin structure is inserted into the membrane and VirG’s passenger domain is secreted through the porin structure.

Native folding occurs at the outer bacterial membrane, with the autochap-erone region comprising residues 591–758 serving as a template and platform for correct folding of the entire passenger domain [82, 85, 86], however partial folding in the periplasm has also been discussed[84]. The autochaperone region has been shown to be essential for folding by mutational analysis [87].

A fragment of VirG comprising the autochaperone region has been crystal-lized by Dr. K. Kühnel (PDB 3ML3, Figure 1.9). The VirG fragment folds into two coils of a right handed parallel β-helix, with the last two antiparallel β-sheets covering the hydrophobic core. β-helical fold is typical for autotrans-porters [82].

VirG localizes to the pole ofShigella, where it recruits factors important for Shigella’s actin-based motility. VirG hijacks the Cdc42-controlled molecular machinery essential for actin assembly. First, IcsA binds to N-WASP and activates it. A ternary complex with Arp2/3 is formed, which stimulates actin assembly and polymerization. N-WASP and the Arp2/3 complex are crucial for Shigella’s ability to move and replicate within the host cell [77].

1.3 Shigella flexneri and Autophagy 15

A B

Figure 1.9: Crystal structure of VirG (591–758)

Side and top view of rainbow-coloured VirG (591–758) from blue (N-terminus) to red (C-(N-terminus)

1.3.4 The secretion protein IcsB–IpgA

IcsB was described first by Allaoui et al. [88] as a virulence factor located on the Shigella virulence plasmid in 1992. Initial studies on a IcsB defective strain indicated, that IcsB was not crucial for invasiveness of bacteria, but caused invading bacteria to be trapped by “protrusions surrounded by two membranes”. This was probably the first observation of IcsB’s role in protect-ing the bacterium from autophagy. IcsB-defective bacteria were therefore not able to spread across cells [88]. IcsB is secreted via Shigella’s TT3S in vivo and in vitro. It is chaperoned by IpgA, the protein originating from the gene downstream of IcsB. The stop codon between the two of them is transient, so that they can be translated and secreted together as a translational fusion protein. IcsB’s middle domain was found to be involved in the interaction [89].

1.3.5 Aims

VirG and IcsB are part of a mechanism exploiting a evolutionary niche by hijacking the host cell’s defense mechanism. In the long term, structural in-sights into the protein structure and their interactions could contribute to the development of specific drugs againstShigellosis.

16 Introduction Hence, the aim of this project was to purify and structurally characterize Shigella flexneri’s proteins VirG and IcsB–IpgA. This involved the develop-ment of a suitable purification strategy for VirG. The VirG passenger domain is a membrane-attached protein, which folds into its native state at the outer membrane of the bacterium. Hence, it would be challenging to find suitable conditions for native folding to occur. Furthermore, protein interactions should be characterized by suitable biochemical methods.