Endothelial cells are specialized cells that line the internal surface of blood vessels and are responsible for the maintenance of vascular permeability. Although serving as a barrier between blood and interstitial fluid, the lung endothelium is composed of a single layer of endothelial cells, making it vulnerable to attack by P. aeruginosa virulence factors. Following disruption of the epithelial barrier, P. aeruginosa virulence factors have access to the endothelium, where proteases and toxins released from P. aeruginosa further disrupt endothelial tight junctions ^[1][62]. As a consequence of dysregulated endothelial cell barriers, P. aeruginosa can migrate into the bloodstream and lead to bacteremia and cause a fatal outcomes ^[2][63]. However, compared to airway epithelium, a small number of studies have investigated the destructive effects of P. aeruginosa virulence factors on lung endothelium ^{[3][4][1][5][6]}[28,50,62,64,65]. Endothelium presents similar yet distinct intercellular junctional components when compared to those of the epithelium. For example, instead of E-cadherin expressed by epithelial AJs, endothelial AJs present VE-cadherin, an endothelial-specific cadherin ^[7][8][66,67]. P. aeruginosa elastase cleaves VE-cadherin ^[9][1][21,62]. Moreover, ExoS and ExoT increase paracellular permeability across endothelial cell monolayers through integrin αvβ5 with activation of RhoA signaling ^[3][10][11][28,68,69]. In addition, compared to the junctional complex in epithelium, the endothelium presents intermingled TJs and AJs ^[12][70]. Interestingly, recent evidence suggests that actin assembly at TJs and AJs are regulated through distinctive mechanisms ^[13][14][71,72]. Lung endothelium and epithelium also share some similar mechanisms in the role of cytoskeleton dynamics in barrier function in response to P. aeruginosa infection. Neural Wiskott–Aldrich syndrome protein (NWASP) plays a critical role in cytoskeleton dynamics and regulates barrier integrity through Rho GTPase signaling and cytoskeletal reorganization in lung endothelial and epithelial cells in response to P. aeruginosa and transforming growth factor beta-1 ^[5][15][64,73]. It has recently been noted that barrier function is more strictly controlled with 10 times higher transendothelial electrical resistance and more developed intercellular junctions in lung microvascular endothelium in comparison to lung macrovascular endothelium ^{[16][17][18][19]}[74,75,76,77]. Additional studies are needed to understand the molecular mechanisms by which P. aeruginosa virulence factors breach the lung microvascular endothelium by modulation of cytoskeletal structures and cytoskeletal regulatory proteins.

2. Cytoskeletal Regulation by P. aeruginosa Virulence Factors

2.1. Regulation of Lung Permeability by Virulence Factors Belonging to

Regulation of Lung Permeability by Virulence Factors Belonging to

P. aeruginosa

Type III Secretion System

Type III secretion system (T3SS) is the major contributor to P. aeruginosa-induced virulence ^{[20][21][22][23]}[78,79,80,81]. Epithelial cells are especially sensitive to the effects of T3SS toxins ^{[24][22][23][25][26]}[25,80,81,82,83]. P. aeruginosa T3SS translocates four exoenzymes (ExoS, ExoT, ExoY, and ExoU) into host cells (Figure 12). These exoenzymes have overlapping, yet distinct pathways to target cytoskeleton components and associated junctional complex, causing cell morphological changes and intercellular junction disruption, leading to a loss of barrier integrity. The interactions of these type III exoenzymes with cytoskeleton components are important in the pathogenesis of P. aeruginosa infection.

Figure 12. Schematic depicting T3SS exoenzymes and their interaction with host intracellular pathways contributing to barrier disruption. These events result in actin stress fiber formation, cytoskeleton rearrangement, and disruption of intercellular junctions, following with increased permeability.

2.2. Regulation of Lung Permeability by

3. Regulation of Lung Permeability by

P. aeruginosa

Secreted Virulence Factors

2.2.1. Elastase

3.1. Elastase

P. aeruginosa elastase (PE) is a secreted metalloproteinase with highly efficient proteolytic activity on a number of host structural proteins in airway epithelium ^{[27][28][29][30][31][32]}[120,121,122,123,124,125]. It has been reported that PE can transiently disintegrate and redistribute tight junction proteins OCLN and ZO-1, induce cleavage of VE-cadherin, and cause actin cytoskeleton reorganization ^{[33][34][1][35][36][37]}[22,23,62,126,127,128]. By using the B.V strain that is known for its high elastase activity, it has been shown that PE is capable of completely degrading ZO-1 and significantly degrading OCLN ^[36][127]. Besides targeting on tight junction proteins, PE has tissue-damaging activities. In addition, PE can degrade lung elastin, an important structural protein for maintaining blood vessel integrity ^[30][38][123,129], as well as matrix proteins including laminin and collagen (type III and type IV), leading to basement membrane impairment ^[39][40][41][130,131,132].

2.2.2. Exotoxin A

3.2. Exotoxin A

P. aeruginosa produces a highly toxic virulence factor exotoxin A (ExoA) which is released into extracellular medium by type 2 secretion system (T2SS) ^[42][43][133,134]. It has ADP-ribosylation activity and affects the protein synthesis processes in host cells. ExoA has been shown to delay wound repair in the animal cutaneious injury model through its effects on cytoskeleton remodeling ^[44][135]. Treatment with ExoA reduces TJs proteins ZO-1 and ZO-2 and increases paracellular permeability in type II pneumocyte cultures ^[34][23]. However, the exact mechanism undergoing ExoA-mediated epithelial barrier damage still need further studies.

23.3. Regulation of Lung Permeability by P. aeruginosa Surface-Bound Virulence Factors

23.3.1. Pilus and Flagellum

Type IV pilus and flagellum are important surface structural components for P. aeruginosa attachment to cell surface and are critical in preparation for T3SS toxin injection ^[45][46][136,137]. Due to the nature as P. aeruginosa surface structure, pilus and flagellum are likely to have roles beyond mediating an initial attachment to the host surface. Evidence show that pilus and flagellum are required for transmigration across epithelial cell junctions ^[45][46][136,137]. Recently, pilus has been shown to preferentially interact with the cell basolateral domain and T3SS effectors are only injected into host cells through their basolateral membrane domain ^[45][46][47][136,137,138]. Internalization of P. aeruginosa in the epithelial basolateral surface requires flagellum binding to heparan sulfate, with subsequent signaling activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinases (PI3K), and protein kinase B (AKT) ^[47][138]. These findings suggest these surface-bound virulence factors may play an important role in mediating P. aeruginosa transmigration through paracellular route.

23.3.2. Lipopolysccharide

Lipopolysccharide (LPS) is a major structure component which is integrated in the P. aeruginosa cell wall and plays an important role in bacterium–host interactions ^[48][139]. LPS is a pro-inflammatory mediator which can increase airway epithelial permeability ^[49][140]. LPS-induced F-actin rearrangement and actin assembly are important for LPS signaling ^[50][59]. However, molecular mechanisms for LPS-induced endothelial cell permeability are still not well understood.

23.4. Regulation of Lung Permeability by Quorum Sensing and Other P. aeruginosa Virulence Factors

Quorum sensing (QS) is a specialized cell density-dependent regulation system in bacteria ^[51][52][53][141,142,143]. These bacterial signals also modulate mammalian airway epithelial cell responses to the pathogen in a process called interkingdom signaling. N-(3-Oxododecanoyl)-L-homoserine lactone (C12) is a small molecule quorum-sensing signal produced by a P. aeruginosa lasR-lasI QS system ^[54][55][144,145]. In addition to the regulation of P. aeruginosa population behavior, C12 also regulates a range of complex biological processes in host cells. In human epithelial Caco-2 cells, C12 induces a decrease in transepithelial electrical resistance (TER), an increase in paracellular flux, a reduction in the expression and distribution of ZO-1 and OCLN, and reorganization of F-actin through activation of p38 and p42/44 pathways ^[56][146]. In intestinal epithelial cells, C12 alters the phosphorylation status of cell junctional components, including E-cadherin, beta-catenin, OCLN, ZO-1, and ZO-3, and JAM-A. In addition, the changes in phosphorylation status of regulatory proteins disrupt the association between junctional components and result in a loss of epithelial barrier and increased paracellular permeability ^[57][58][24,147]. C12 also induces degradation and de-location of TJs proteins (OCLN and tricellulin) in intestinal epithelial Caco-2 cells ^[59][148]. These findings collectively indicate that C12 induces epithelial paracellular permeability possibly through a mechanism that mediates the disassembly of intercellular links. C12 induces myofibroblast differentiation in vitro and in vivo for accelerated wound healing ^[60][149]. In cultured nonpolarized airway epithelial cells, C12 induces massive morphological changes of cell structure with perturbed gap junction shortly after application ^[61][150]. C12 may also facilitate dissemination of virus into bloodstream ^[62][151].

Rhamnolipids

P. aeruginosa produces biosurfactants called rhamnolipids ^[63][64][152,153]. Rhamnolipids act as a potent detergent and have been reported to disrupt intercellular junctions in sheep tracheal epithelium at high concentrations ^[65][154]. Rhamnolipids induce ciliostasis of airway epithelial cells and may disrupt their barrier function, allowing invasion of pseudomonas ^[66][12]. Alzheimer’s disease (AD) has been attributed to chronic bacterial infections, and the levels of rhamnolipids in sera and cerebrospinal fluid of AD patients are significantly increased when compared to controls ^[67][155]. However, the meaning of the increased rhamnolipids levels in AD patients and AD pathogenesis is unclear so far.