CLINICAL MANIFESTATIONS, COMPLICATIONS AND TRANSMISSION 

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Shigella invasion routes of the intestinal epithelium

The initial routes of invasion are not entirely clear and will be further discussed in detail. Early observation of biopsies from Shigella-infected patients suggested the gut-associated lymphoid tissue as one of the primary sites targeted (Mathan and Mathan 1991). In the rabbit ileal loop model, M cells were reported as the initial route of entry (Wassef, Keren, and Mailloux 1989). However, a guinea pig model that recapitulates several clinical features of the human infection shows that epithelial cells are invaded as early as 2-6 hours post-administration (Shim et al. 2007). Using a guinea pig intra-rectal inoculation model, it was observed that enterocytes at the colonic crypts mouths were targeted at the onset of colonization and bacteria spread by cell-to-cell transmission into the crypts along the infection (Arena et al. 2015). In the rabbit ileal loop model, there is evidence that Shigella traverses the cell monolayer through M cells (Sansonetti et al. 1996), a step that would allow it to infect macrophages in the M cell pouch. According to this model, after inducing the pyroptotic death of macrophages, Shigella would invade epithelial cells from the basal side. Pyroptosis also leads to the release of pro-inflammatory cytokines IL-1β and IL-18, and invasion of epithelial release of IL-8. This causes a strong inflammatory response that recruits polymorphonuclear leukocytes and destabilizes the intestinal epithelium, hence facilitating invasion from lumenal bacteria.

The Shigella Type III Secretion System

The Shigella T3SS structure

A key element for the pathogenesis of Shigella is its T3SS. This apparatus functions as a needle, translocating effector proteins from the bacterial cytoplasm to the host cell cytosol (Figure 5). Assembly of fully functional T3SS apparatus is regulated by temperature but the injection of T3 effectors is dependent on host membrane contact (Veenendaal et al. 2007).
The T3SS is generally divided in three parts: the basal body, the needle and the tip complex. The basal body anchors the apparatus to the bacterial membranes (Figure 5). MxiD and MxiM form a ring in the outer membrane. On the cytoplasmic side, a C-ring formed by the proteins Spa33, MxiK and MxiN mediates the recognition, sorting and secretion of effectors, where the ATPase activity of Spa47 is involved (B. Hu et al. 2015; Burgess et al. 2016; Morita-Ishihara et al. 2006). Spa32 interacts with Spa40 and controls the length of the needle (Botteaux et al. 2010; Tamano et al. 2002). Other proteins are also present, but their roles remain unclear.
The needle is composed of the major MxiH and the minor MxiI subunits. MxiH assembles in a helical fashion and constitutes the needle rod with a 2-3 nm inner diameter (Blocker et al. 2001), while MxiI is proposed to be involved, together with MxiC, in the secretion activation upon contact of the host membrane (El Hajjami et al. 2018; Cherradi et al. 2013).
Before contact with the host membrane the needle tip complex composed of IpaD and perhaps IpaB remains in a closed conformation (Veenendaal et al. 2007; Cheung et al. 2015). IpaB and IpaC are translocated upon contact with host cells, likely by interacting with membrane enriched in cholesterol and sphingolipids-containing membranes (Goot et al. 2004). IpaB and IpaC form the translocon allowing the translocation of effector proteins.
Figure 5. Structure of the Shigella T3SS. Tip components shown in yellow and green. Needle components in blue-greens, transmembrane base dark purple, inner membrane export apparatus light purple, C-ring and ATPase complex in light blues. OM, outer membrane. IM, inner membrane
Figure 5. Structure of the Shigella T3SS

Shigella T3SS effector proteins

At 37°C the transcriptional activator VirF is expressed and activates virB expression. VirB is also a transcriptional activator that activates the ipa (Invasion Plasmid Antigens) operon and the mxi (Membrane Expression of Ipa) – spa (Surface Presentation of Ipa) operon. Expression of these operons triggers bacterial entry mediated by the T3SS (Du et al. 2016), along with other effector genes. Injection of effector proteins is temporarily divided into two waves. After initial host cell contact, the injection of the first wave of effectors happens within minutes (Enninga et al. 2005). Shigella T3SS uses chaperons to prevent degradation and to target effector proteins to the secretion apparatus. The IpgC chaperone also regulates the expression of the second wave of T3S effectors. Prior to activation of T3 secretion, the IpgC chaperone associates with IpaB and IpaC and prevents their premature interaction in the bacterial cytosol (Parsot 2003; Pilonieta and Munson 2008). After activation of T3 secretion, IpaB and IpaC are secreted, releasing IpgC, which may then interact with the MxiE transcriptional activator. This allows the formation of the complex IpgC-MxiE that promotes triggering the transcription of genes harboring a MxiE box, (Figure 6) corresponding to genes of the second wave of effectors (Kane et al. 2002). Genes belonging to the second wave of effectors are mainly involved in high-jacking host signaling, apoptosis inhibition and regulation of long-term effects on the host immune response (For review, see Mattock and Blocker 2017).

Main players of the cytoskeleton

Actin monomers

The actin cytoskeleton is commonly targeted by pathogens, being highly conserved among animals and a central element in various cellular processes. Actin monomer, termed G-actin, can polymerize into actin filaments, or F-actin. ATP-G-actin polymerizes into ATP-F-actin, which has an intrinsic ATPase activity leading to ADP F-actin. ADP F-actin is less stable than ATP G-actin, rendering ADP F-actin filaments prone to disassembly (Dominguez and Holmes 2011).
The incorporation of actin monomers is asymmetrical. The barbed end (or plus end) of actin filaments incorporates ATP-actin monomers, while the pointed end (or minus end) is more prone to be disassembled (Figure 7A). Simultaneous incorporation of monomers at one end and disassembly on the other gives rise to treadmilling.

Actin sequestering proteins

In the intracellular milieu, actin binds to profilin. This complex represents the main source of monomers for polymerization. Profilin catalyzes the ADP/ATP exchange in actin monomers, while inhibiting the spontaneous filament nucleation property of actin monomers (Dominguez and Holmes 2011). Profilin-actin also binds to other proteins involved in actin polymerization, such as formins and Ena/VASP proteins (Kovar et al. 2006) (Figure 7).
Thymosin-β4 is a peptide of 43 residues. In its N-terminus it has short helix that binds the actin barbed end and the rest of the helix, forms an extended region that binds the front surface of actin and a second helix that caps actin pointed end (Xue et al. 2014). This results in the blockage of engagement of actin monomers into polymerization. However, profilin and thymosin-β4 are constantly exchanging actin monomers, allowing elongation of actin filaments (Pantaloni and Carlier 1993).

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Severing proteins

Depolymerization in cells is enhanced by proteins of the ADF/Cofilin family, which includes Actin Depolymerization Factor (ADF), cofilin-1, cofilin-2 and twinfilin. This family of proteins have a higher affinity for ADP-actin than for ATP-actin, and therefore bind to ADP-actin fiber stretches and promote the severing of these filaments (Barbara W. Bernstein 2010).

Actin nucleators

Actin nucleators initiate the de novo polymerization of actin filaments. The Arp2/3 complex is one of the best characterized actin nucleators. It is composed of a core of 7 proteins. Upon the interaction of Nucleation Promoting Factors (members of the Wiskott-Aldrich Syndrome Protein family, WAVE, among others), actin filaments and actin monomers, Arp2/3 is recruited and activated by Arf1 and Rac1 (Koronakis et al. 2011). Through the VCA domains of the Nucleation Promoting Factors, Arp2/3 is connected to the mother actin filament and nucleates the polymerization of a new branched actin filament (Pollard 2007). Given the Arp2/3 structure, the daughter filament elongates at an angle of 70° from the mother filament (Amann and Pollard 2001). At a large scale, polymerization of new branched filaments upon existing filaments give rise to branched actin networks, which are enriched in lamellipodia (Figure 7B).
In opposition to Arp2/3, formins nucleate and elongate unbranched actin filaments in filopodia, stress fibers and actin cables. They are characterized by the presence of the Formin Homology Domains 1 and 2, which mediate their interaction with profilin and their nucleation activity, respectively. Their ATP-dependent activity results in a highly processive actin polymerization (Romero et al. 2004). In a similar fashion, Ena/VASP proteins also promote the nucleation and elongation of actin filaments (Figure 7C), and works in neurons show that they are particularly important for filopodial formation (Lebrand et al. 2004).

Capping proteins

Capping protein and CapZ bind to the barbed end of actin filaments, blocking both the addition and loss of actin subunits (Isenberg, Aebi, and Pollard 1980; J. Xu, Casella, and Pollard 1999), therefore preventing the assembly/disassembly of filaments. In conjunction with the branching activity of Arp2/3, a network of short and highly branched actin filaments can be formed (Figure 7B) (M. Edwards et al. 2014).

Cross-linking proteins

Individual actin filaments can be bundled together through the action of actin cross-linkers, such as α-actinin isoforms 1 and 4 or fimbrin. These proteins contain Actin-Binding Domains (ABD) that connects two separate actin fibers. α-actinin has also the ability to associate with other cytoskeletal, signaling and membrane molecules (Sjöblom, Salmazo, and Djinović-Carugo 2008).
The organization into bundles by filament bundling facilitates the mechanical contraction exerted by myosins molecular motors. In particular, Myosin II transforms the chemical energy released by ATP hydrolysis into mechanical work that results in pulling of actin filaments (Figure 8). The pulling activity is the result of the myosin II polymerization into bipolar filaments, with the motor domains walking in opposing cross-linked actin filaments (Figure 8) (Sweeney and Holzbaur 2018).
Figure 8. Myosin II and α-actinin in actin filament contraction. Myosin II polymerizes into bipolar filaments, with the motor domains walking in opposing actin filaments cross-linked by α-actinin. Modified from (Svitkina 2018).

Adhesion structures

Integrins

The intracellular actomyosin cytoskeleton dynamically interacts with the extracellular environment through linker molecules anchoring transmembrane receptors that connect with the extracellular environment. Integrins and cadherins are two of the main receptors that connect the actomyosin cytoskeleton with the outer environment and to respond to mechanical and biochemical cues.
Integrins are cell adhesion receptors, and its super-family is composed of 18 types of α subunits and 8 types of β subunits, which can associate into 24 different αβ heterodimers, the β subunit determining the family. The α and β subunits are non-covalently associated transmembrane proteins. Different pairs can bind to different ligands, such as RGD motif (present in fibronectin and vitronectin), collagen, laminin, or ligands in leukocytes (Kechagia, Ivaska, and Roca-Cusachs 2019; Hynes 2002) (Figure 9).
Although not proven for all αβ pairs, some integrins can transition from a “bent closed” or inactive form, to an extended closed and finally to an extended open. This transition is associated with an increase in the affinity of the integrin for its substrate.

Table of contents :

1 INTRODUCTION 
1.1 BACILLARY DYSENTERY
1.1.1 CLINICAL MANIFESTATIONS, COMPLICATIONS AND TRANSMISSION
1.1.2 EPIDEMIOLOGY
1.2 SHIGELLA SPP.
1.2.1 SHIGELLA CLASSIFICATION
1.2.2 EVOLUTION OF SHIGELLA
1.2.3 SHIGELLA VIRULENCE PLASMID
1.3 OVERVIEW OF SHIGELLA INVASION
1.3.1 ANATOMY OF THE INTESTINAL EPITHELIUM
1.3.2 SHIGELLA INVASION ROUTES OF THE INTESTINAL EPITHELIUM
1.4 THE SHIGELLA TYPE III SECRETION SYSTEM
1.4.1 THE SHIGELLA T3SS STRUCTURE
1.4.2 SHIGELLA T3SS EFFECTOR PROTEINS
1.5 MAIN PLAYERS OF THE CYTOSKELETON
1.5.1 ACTIN MONOMERS
1.5.2 ACTIN SEQUESTERING PROTEINS
1.5.3 SEVERING PROTEINS
1.5.4 ACTIN NUCLEATORS
1.5.5 CAPPING PROTEINS
1.5.6 CROSS-LINKING PROTEINS
1.6 ADHESION STRUCTURES
1.6.1 INTEGRINS
1.6.2 ADHESION FORMATION AND MATURATION
1.6.3 TALIN AND VINCULIN MOLECULAR CLUTCH
1.6.4 FORCE GENERATION AND MATURATION
1.7 ACTIN CYTOSKELETON REORGANIZATION BY BACTERIAL PATHOGENS
1.7.1 BACTERIAL ADHESION AND INTERNALIZATION STRATEGIES
1.7.2 E. COLI FIMH CATCH-BOND ADHESION
1.7.3 ZIPPERING: LISTERIA MONOCYTOGENES INVASION MECHANISM
1.7.4 ATTACHING AND EFFACING LESION PATHOGENS
1.7.5 SALMONELLA “TRIGGERED” INTERNALIZATION
1.7.6 SHIGELLA INVASION AND CYTOSKELETON REMODELLING
1.8 IPAA
1.8.1 IPAA VINCULIN BINDING SITES
2 RATIONALE OF THE PHD PROJECT 
3 ARTICLE 1
3.1 OVERVIEW
4.1 OVERVIEW
5 ARTICLE 3
5.1 OVERVIEW
6 DISCUSSION 
6.1 IPAA VBS3 BINDING TO TALIN
6.2 VINCULIN SUPRA-ACTIVATION: A NOVEL INTERACTION MODE
6.3 FUNCTIONAL IMPLICATIONS OF VINCULIN SUPRA-ACTIVATION ON CELL ADHESION
6.4 IPAA VBSS ROLE DURING SHIGELLA INVASION
6.5 GENERAL CONCLUSION
7 REFERENCES

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