Mol Biol Rep (2014) 41:6591–6610 DOI 10.1007/s11033-014-3543-5

The role of epithelial tight junctions involved in pathogen infections Ru-Yi Lu • Wan-Xi Yang • Yan-Jun Hu

Received: 20 February 2014 / Accepted: 20 June 2014 / Published online: 26 June 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Tight junctions (TJs) are sealing complexes between adjacent epithelial cells, functioning by controlling paracellular passage and maintaining cell polarity. These functions of TJs are primarily based on structural integrity as well as dynamic regulatory balance, indicating plasticity of TJ in response to external stimuli. An indispensable role of TJs involved in pathogen infection has been widely demonstrated since disruption of TJs leads to a distinct increase in paracellular permeability and polarity defects which facilitate viral or bacterial entry and spread. In addition to pathological changes in TJ integrity, TJ proteins such as occludin and claudins can either function as receptors for pathogen entry or interact with viral/bacterial effector molecules as an essential step for characterizing an infective stage. This suggests a more complicated role for TJ itself and especially specific TJ components. Thus, this review surveys the role of the epithelial TJs involved in various pathogen infections, and extends TJ targeted therapeutic and pharmacological application prospects. Keywords Tight junction  Claudin occludin  Paracellular permeability infection

R.-Y. Lu  Y.-J. Hu (&) Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China e-mail: [email protected] R.-Y. Lu  W.-X. Yang (&) The Sperm Laboratory, Institute of Cell and Developmental Biology, College of Life Sciences, Zhejiang University, Hangzhou 310058, China e-mail: [email protected]

Introduction Tight junctions (TJs) are sealing complexes at the most apical position of the lateral membrane interface of adjacent epithelial cells [1]. Several studies revealed that TJs consist of three families of proteins: claudins, TJ-associated MARVEL proteins [(TAMPs) occludin, tricellulin, and MarvelD3], and the junction adhesion molecule (JAM) [2]. Upon multiple interactions of these protein components, TJs are maintained in a dynamic balance by numerous regulatory effects. During assembly and disassembly of TJ, phosphorylation and dephosphorylation of TJ proteins such as occludin and claudin are of great value. Based on the dynamic structure, TJs play a significant part as a barrier of epithelial cells [3–5], via selectively controlling the paracellular passage of solutes and water as well as microorganisms and toxins. In addition, TJs regulates the cellular polarity by prohibiting the intramembrane distribution of proteins and macromolecules between the apical and basolateral membrane domain. Given the indispensable functions for epithelial cells, a growing number of bacteria and viruses are reported to infect the human body partly depending on TJ disruption. Altered TJ structure by up/down-regulated expression of TJ proteins, redistribution, and disruption of protein–protein interactions have been observed in distinct states of various infections, along with barrier breakdown and loss of cell polarity. This may lead subsequently to more severe diseases. Besides, TJ components are demonstrated to intimately interact with viral and bacterial infection as an essential step. For instance, claudin-1 and occludin are taken as coreceptors during the HCV entry [6]; JAM is a receptor for reovirus [7]. Considering the interactions, new attempts to exploit TJ and TJ-associated proteins for therapeutic interventions or drug transportation are carried out.

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Fig. 1 Structure of tight junctions and dynamic assembly and disassembly regulation. Tight junctions (TJs) are sealing complexes located at the most apical side of lateral membrane of adjacent epithelial cells. Claudins, occludin and JAM are three transmembrane protein families, which interact with scaffolding proteins via PDZ binding motifs. ZOs connect TJ proteins with actin cytoskeleton as a

bridge and in this way TJ architecture and function can be dynamically regulated. During assembly and disassembly, TJs undergo endocytosis, migration and recycling. There are multiple cytokines and kinases listed which have a great regulatory effect on TJ proteins, especially occludin and claudin, and subsequently influence the integrity of TJ

However, a lot of detailed mechanisms remain unclear: which component of TJs participates in the infection? Which stage of infection requires the involvement of TJ and how are TJ proteins interacting with the pathogens and influence the life cycle of bacteria and viruses? In this review, pathogen infections such as hepatitis viruses, gastrointestinal bacteria, and respiratory tract infective viruses as well as female reproductive infections will be primarily focused on and the role of TJs in association with harmful pathogens will be highlighted. With more detailed investigations, TJ targeted pharmacological and therapeutic application may make great progress and will be applied in clinical therapy for diseases elicited by viruses and bacteria.

and MarvelD3) and JAM. Recently claudins and TAMPs are classified on the basis of their permeability attribute into sealing, channel-forming, inconsistent and unknown function proteins [2]. In contrast to these transmembrane proteins mentioned above, there are peripheral membrane proteins that can attach these proteins to cytoskeleton and initiate signaling pathways to regulate TJ, such as zonula occludens (ZOs), MAGI proteins, and cingulin [5, 9]. In the form of belt-strands, TJs function as a barrier to control the paracellular passage and selectively allow solutes and water as well as some microorganisms to get through. Thereby, when the barrier breaks down, bacteria and viruses existing in the lumina can easily enter the paracellular cavity and replicate in the internal environment. In addition, TJs regulate the cellular polarity via inhibiting the intramembrane distribution of proteins and macromolecules between the apical and basolateral membrane domain. The cellular polarity is reflected by a polarized division of the cell membrane into the apical and basolateral domains and in return some distinct polarity complexes have a crucial effect on the location of TJ and maintain the cellular balance [10]. Furthermore, primary cilia have also been demonstrated to play a role in maintaining cellular polarity. Nevertheless, Di Mo’ s experiment showed that in response to nucleofection in MDCK cells the cell polarity was decreased as a result of

TJ structure and function TJs are intercellular structures commonly seen in the mucosal epithelia of the stomach, intestine, gall bladder, uterus, and oviduct [1]. In freeze-fracture electron microscopy, TJs are shown as rows of closely spaced proteinaceous attachment particles, maintaining the opposing cell membranes close to each other (see Fig. 1) [8]. According to genetic or molecular kinship, TJ proteins are classified into: claudins, TAMPs (occludin, tricellulin,

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compromised TJ fence function, while the cilia frequency and length were not altered, suggesting it had little relation to cilia for TJs to maintain cellular polarity [11]. Although the two functions are built on the same specific architecture of TJ, they can be distinguished from each other when responding to external stimulants. Therefore, it is necessary to figure out the exact effect on TJ that the infections elicit.

TJ proteins Starting from the early 1990s, proteins involved in the constitution of TJs have been identified. The first member of the family, occludin, was described by Furuse et al. [12] in 1993 and since then the field has witnessed a wealth of new information. Occludin is a tetra-transmembrane protein, consisting of 504 amino acids, with four transmembrane domains, two extracellular loops (ECLs), generally only expressed in cells that form TJs [13]. The intracellular carboxyl terminal part (50 % of the sequence) has several potential sites that can interact with ZO-1, ZO-2 [5], as well as phosphorylation sites [14], while the functional significance of the ECLs is uncertain as yet. Researches on occludin demonstrate that multiple domains of occludin take part in the modulation of paracellular permeability [15, 16], and the translocation of tricellulin to tricellular TJs [17]. However, deletion of occludin does not affect the constitution of TJs revealed in occludin-deficient embryonic stem cells [18]. Together with these analyses, occludin is supposed to have a regulatory effect on TJ but does not represent an essential component for TJ formation. Generally speaking, occludin is a highly-conserved TJ protein among species and different tissues, with the only variant termed occludin 1B, whose patterns of phosphorylation differ to those of occludin [19]. Tricellulin (MarvelD2) and MarvelD3 are typical tetraspanin members of TJ components and they constitute the TJ-associated marvel proteins together with occludin, which contain a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link [20]) domain. Tricellulin is the only known protein that is highly concentrated at tricellular TJs [21]. Nayak’s [22] experiment in the mice with a mutated variant of tricellulin revealed disruption of TJ architecture, which resulted in toxicity to the cochlear hair cells in the inner ear epithelia, indicating a compromised epithelial barrier function in the absence of tricellulin. Another important component of TJ is the claudins family, which in mammals composes of 27 members with molecular weights between 20 and 27 kDa [13, 23, 24]. Similar to occludin, claudins contain four transmembrane domains, two extracellular (ECL) and one intracellular

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loop (ICL), but can be characterized by a differential tissue and cell type expression pattern [24–26]. The function of the crucial TJ components is principally dependent on their two ECLs. ECL1 primarily contributes to the paracellular barrier or selective channel attributes while ECL2 by comparison maintains the opposing lateral cell membranes in narrow contact [26]. Most of claudins contain C-terminal PDZ binding motifs, through which they directly bind with intracellular proteins, including ZO-1, ZO-2, and ZO-3. To conclude, claudins are pivotal components in the structure and function of TJs and supposed to be the ‘‘backbone’’ of the TJ barrier. Furthermore, recent studies have revealed the role of claudins involved in some hereditary diseases and cancers [27, 28], which can also provide a new sight into the crucial role of claudins in TJ. In addition, there are some types of proteins which are not necessary for TJ but generally intimate with TJ proteins. JAMs, as members of the immunoglobulin superfamily, are revealed as glycosylated transmembrane proteins [27]. These transmembrane domain proteins are demonstrated to play an important role in early stages of TJ formation and regulation. The intracellular constituents of TJs, ZO-1, ZO-2 and ZO-3 are membrane-associated guanylate kinase proteins consisting of a PSD95/Dlg/ZO-1 domain, an SH3 domain, a guanylate kinase domain, an acidic domain and an actin-binding region [29]. Via the PDZ motif, they connect with the C-terminal cytoplasmic domain of occludin and claudins and interact with the actin cytoskeleton as well [29–31]. Thus, ZOs are suggested to be the bridge between cytoskeleton and transmembrane proteins. Besides, ZO-1 and ZO-2 can decide where the polymerization of claudins takes place, indicating their indispensible role towards TJ formation [32]. Furthermore, ZO-1 and ZO-2 are both members of a group of proteins named NACos, for proteins that can localize to the Nucleus and Adhesion Complexes [33]. ZO-1 sequesters ZONAB (a transcription factor) and thereby regulates cell density and proliferation, whereas for ZO-2 the association with Scaffold attachment factor-B [34], activator protein-1 [35] and a repressive function on b-catenin transcriptional activity [36] were reported. They are playing a role in a TJ-associated signal-transduction pathway which regulates nuclear gene transcription.

TJ dynamic assembly and disassembly regulation TJs go through assembly and disassembly in a dynamic pattern in every epithelium, which is a continuous process from cellular polarization, recruitment of TJ proteins, stablization (via interactions with the actin cytoskeleton), maintenance (via membrane trafficking) and to disassembly (via ubiquitination events) [2, 10]. Thus all these

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closely related proteins, molecules and cytokines involved in these processes have to be considered to have a regulatory effect. For instance, Cdc42 is crucial to decide whether the endocytosed TJ proteins recycle to the membrane or are translocated to lysosomes for degradation [10]. Besides, the interactions of TJ proteins as well as the connection with the actomyosin cytoskeleton determine the precise TJ structure and location during assembly, indicating an indispensible role for modification of TJ proteins (see Fig. 1). Among the multiple proteins, occludin is the one reported to be greatly related to regulatory function. Phosphorylation and dephosphorylation of occludin is a critical post-translational modification. Multiple kinases and phosphatases have been characterized for occludin, such as PKC, PKA, MAPK, PI3K, CK1, CK2, tyrosine kinases, and RhoK [14, 37], with different effects. In addition, some specific sites targeted by these kinases have also been spotted. Within occludin C-terminal domain there is a hotspot between Y398 and S408 that can be phosphorylated by PKCg and f, Src kinase and CK2 together [14], indicating a cooperative way to maintain occludin function. In fact, the mobility of occludin is not only determined by different kinases but also by the site and the degree of phosphorylation that largely defines the localization and the function of the molecule. For instance, more highly phosphorylated occludin usually affects the epithelial permeability to a higher degree [13]; phosphorylation at tyrosine destabilizes TJs while at threonine by PKCs promotes the TJ assembly [14]. Dependent on the phosphorylation pattern, the interactions of TJ proteins are also modulated. Previous experiments showed that CK2mediated phosphorylation of Occludin S408 altered interactions between occludin, ZO-1, claudin-1, and claudin-2, and led to regulation of functional properties of TJ [37, 38]. In addition to direct phosphorylation, occludin is involved in the cytokine-induced pathway leading to alterations in TJ architecture and function. Van Itallie et al. [39] indicated that occludin expression level affected cytokine IFN c and TNF a induced barrier function. Moreover, Marchiando et al. [40] have demonstrated that occludin endocytosis is essential in the TNF-induced barrier dysfunction which is dependent on caveolin. Thereby, we can see a cluster of regulatory effects on the TJ structure and function where occludin plays a crucial but complicated role. In addition to occludin, there are several phosphorylation sites on the carboxyterminal tail of claudins as well, related to PKC, PKA, MAPK, WNK, myosin light chain kinase (MLCK), c-Src, RhoK, and the ephrin receptor family [2, 13]. However, currently only a few protein kinases have been confirmed to be involved in TJ regulation and more investigations are needed here. Moreover, palmitoylation has also been described to be a potent regulatory way by modulating the claudin-14 localization and forming efficient TJ barrier

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functions [41]. Specific sites in claudin-1 termed as Yin Yang sites have been identified to be competitively modified by phosphorylation and O-glycosylation, which are involved in protein stability and TJ function [42]. As stated above, TJ functions provide a barrier and maintain cellular polarity. Proteins that regulate cell polarity may have an effect on the maintenance of TJ structure in turn. For example, the suppression of PALS1 and PATJ resulted in delayed formation of TJ and reduced TER which is suggested to be mediated by a failure of recruitment of Par6/Par3/aPKC complex to TJ [43]. A similar effect caused by CRB3 related to cell polarity has been reported too [5]. Several studies have indicated that the TJ dynamics interacted with actin remodelling and actomyosin contraction involving clusters of downstream signaling pathways [44]. To summarize, assembly and disassembly of TJs takes place continually and circularly in epithelial cells, during which TJs undergo remodeling including the endocytosis, migration and recycling of components. The dynamic process is regulated both from intracellular and extracellular events. Intracellular events include energy depletion and changes in cAMP levels. Extracellular events mainly include: direct interactions to other cell proteins or external antigens; indirect paracellular (cytokine) effects [45] and endocrine stimuli; oxidative stress and calcium level imbalance [46]. For instance, Meprin A cleaves the TJ protein occludin, which leads to disassembly of TJ and disruption of epithelial barriers [47]. Additionally, cytokine factors such as TGF-b3 is demonstrated to have an up-or down-regulatory effect on genes encoding TJ proteins with increased or reduced permeability in cultured epithelial cells over a time course [48], while TNF-a can induce increased TJ permeability in Caco-2 monolayers by activation of the ERK1/2 signaling pathway [49]. Early studies have revealed intracellular calcium is attached to TJ reassembly after ATP depletion-repletion by altering localization of occludin and ZO-1 [50].

Altered TJs in pathogen infections TJs are dynamic systems both at the structural and molecular level, which is indeed important for a correct development of their functions. The barrier and fence functions of TJs are critical for epithelial cells to be divided into distinct tissue compartments and keep homeostasis. Thus, it is widely suggested that there may be an intimate interaction in TJ alteration and various infections and diseases, as shown by a number of articles. For instance, in patients with ulcerative colitis [51] or Crohn’s disease [52], perturbation of TJs and barrier dysfunction have been observed. Following our studies, TJs and TJ components

EspF, Map, EspI, EspG, CNF-1, Tir

EPEC

Occludin, claudin-1, ZO-1

Claudin-1, occludin, claudin-6, claudin-9

CagA, VacA

Fiber

H. pylori

Adenovirus Claudin-4, occludin, ZO-1

Claudin-2, occludin, ZO-1, claudin-7

HIV-1

CAR

ZO-1, JAM, claudin-4, claudin-5, occludin

RSV

Glycoprotein gp120

CPE

Clostridium perfringens

Claudin-3, claudin-4, claudin-1, occludin

Occludin, claudin-1 ZO-1, claudin-1, occludin

STb

EAEC

ETEC

ZO-1, occludin, claudin-2, claudin-3

Glycoproteins E1 and E2

HCV

Involved TJ component

EHEC

Effector molecules

Pathogen

TNF-a

TGF-b1/PKC d/HIF-1 a/NF-jB

ROCK, MLCK, urease, PKC

TNF, PI3K/Akt

RhoK, PI3 K, MLCK

PKA

Involved cytokines and kinases

Table 1 Cytokines and kinases involved in pathogen infections, as well as changes in TJ

Proteins expression and mRNA level of claudin-2, occludin and ZO-1;; increased permeability

Expression of claudin-4, occludin and ZO-1:; gene expression of claudin-2, -4, -7, -9, -14, and -19, occludin, ZO-2:

CAR–CAR self-interaction;; TER reduction

Redistribution of ZO-1 and JAM; actin rearrangement; cell polarity loss; TJ disruption

Formation of CPE complexes, TJ disruption

Redistribution and/or fragmentation of ZO-1, claudin-1, and occludin

Redistribution of occludin and claudin-1

Redistribution of occludin and claudin-3; claudin-2 expression:; mRNA levels of occludin and claudin-2, claudin-3:

Redistribution of TJ proteins; claudin-1occludin and occludin-ZO-1 interactions;; ectopic TJ strands; a shift in phosphorylated occludin

Redistribution of claudin-1, occludin and ZO-1

Changes in TJ

[164, 165, 167]

[158]

[146, 150, 153]

[127, 130, 131, 140–142]

[112, 114, 115, 119–121]

[110]

[108]

[100, 102, 103]

[78, 79, 92, 94, 96]

[59, 60, 62, 70, 74, 77]

Reference

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can be modulated or changed in their structural and functional patterns during pathogen infections (see Table 1). In the following discussions we primarily investigate the interactions between various viral or bacterial infections and TJs, and explore pathogen induced alterations of TJ based on a cellular, molecular and even gene level.

Hepatitis C virus infection HCV entry requires glycoprotein-receptor interactions Hepatitis C virus (HCV) infection is a central cause of severe hepatocellular carcinoma and cirrhosis, being one of the most common viral infections worldwide [53]. HCV in primary targets hepatocytes and can induce a systematic disease although the pathogenicity is not fully understood. Multiple articles have demonstrated that HCV attachment and entry into host cells is a highly coordinated and intricate process, during which the viral envelope glycoproteins

Fig. 2 The role of TJs involved in the infections of HCV, EPEC and Clostridium perfringens. TJs play a crucial role in these pathogen infections. During HCV infection, claudin-1 and occludin are indispensable factors which are associated with the viral envelope glycoproteins E1 and E2, and trigger viral entry to the epithelial cells. After clathrin-mediated endocytosis, claudin-1 and occludin also engage in the fusion process and cell–cell transmission. During EPEC infection, a pedestal of polymerized actin directly forms beneath the attachment and various effector molecules are secreted into host cytoplasm to exert multiple effects. Intimin-Tir interaction is involved

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E1 and E2 interact with host factors (see Fig. 2). In vivo, HCV invades the liver through the sinusoidal blood, and primarily contacts the basolateral surface of hepatocytes. Low-density lipoprotein receptor [54] and cell surface glycosaminoglycans, including heparan sulfate [55] are involved in the early step of HCV attachment and thus virus may aggregate to interact with other host factors initiating viral entry [56], identified as tetraspanin CD81 [57], the scavenger receptor class B type I (SR-BI) [58], the TJ-associated proteins claudin-1 [59] and occludin [60]. Experiments that reveal co-expression of these four host factors can render human nonliver cells or murine cells infectable with HCV implicate that they constitute the minimal viral receptor requirement [60]. Claudin-1 is required as a receptor, in association with CD81 Claudin-1, a crucial member of TJ proteins, has been identified as a receptor for HCV entry as mentioned above. Krieger and

in pedestal formation, PLC-g1 phosphorylation and PI3K activation; EspF leads to actin rearrangement; Map targets mitochondrial events; EspI inhibits proteins trafficking from ER to Golgi; EspG disrupts microtubules (MT) with Orf3. During Clostridium perfringens infection, CPE binds with receptors claudin-3 and claudin-4 and changes into a prepore complex as CH-1. With the presence of occludin, CH-1 turns into CH-2 and forms a transmembrane pore for Ca2? influx. The increasing Ca2? level causes apoptosis or oncosis, TJ disruption and cell death

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co-workers revealed that claudin-1 and CD81 act as receptors in a similar pattern and anti-CLDN1 antibodies effectively inhibit the interaction as well as envelope glycoprotein E2 association with the cell surface [61], suggesting a cooperative manner of claudin-1 and CD81 in HCV entry. Interestingly, claudin-6 and claudin-9 are also identified as cofactors in the HCV entry; but the experiment suggested claudin-6 and -9 could not initiate HCV entry effectively while claudin-1 expression was blocked in most cell lines [62]. Following initial attachment and binding, HCV enters into cells by clathrin-mediated endocytosis [63] and clathrin-coated vesicles are delivered to early endosomes to undergo cell fusion triggered by acidic pH [64]. Besides, CD81 is believed to form complexes with claudin-1 [65] and both get coendocytosed via a clathrin-and dynamindependent process [66]. This can be promoted by HCV infection, supporting a role of claudin-1 and CD81 trafficking in virus internalization. In addition to that, Evans’s experiments have identified that claudin-1 also engages in the HCVgp-mediated membrane fusion and is required for a late step in HCV entry [59]. Prior to that, a sequence of events including CD81 activity is suggested to occur and induce downstream effects to enable interactions of claudin-ECL1 to virion. However, the mechanism whether claudin-1 directly joins in the fusion process or indirectly gets involved in it by triggering a series of signaling pathways to make conformational changes on the membrane remains unknown. Moreover, there is a time lag between HCV internalization and fusion reported by Meertens et al. [64] which indicates additional events which may be related to trafficking receptors. Claudin-1 is located both on the apical and basolateral surfaces in normal liver tissue but only the expression on the basolateral membrane increases during viral infection [67]. Additionally, Evans and colleagues have revealed HCVpp (pseudoviral particles) susceptibility is determined by residues in the first ECL of claudin-1 [59], rather than the C-terminal intracellular tail and the putative palmitoylation sites. These observations imply that claudin-1 firstly taken as the receptor for the HCV entry has little relation to TJ. Nevertheless, a study has suggested that CD81 can mediate the lateral migration for the virus to the TJs for subsequent infection via Rho GTPase familymediated actin rearrangement [68]. This discovery keeps consistent with our previous idea that the virus first contacts with the basolateral surfaces, but also provides a possible pathway that can be exploited by the virus to move to where TJs are located via actin cytoskeleton dynamics. It may account for the observation that both the TJ-associated proteins on the basolateral site and the TJs are involved in the HCV infection. However, due to different cell lines and viral system applied in experiments, there are still seemingly controversial conclusions that need to be settled.

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Occludin is required for a postbinding step Occludin, another crucial TJ associated protein, is also described as a host receptor probably for a postbinding step during the viral entry. Occludin knockdown by shRNA transduction evidently lowered the susceptibility of Huh7 and PLC/PRF/5cells to HCVcc infection compared to controls, and cell–cell fusion experiments demonstrated occludin was involved in a HCVgp-dependent cell fusion, not the replication or initial attachment [69]. As stated above claudin-1 is also necessary for the cell–cell fusion, we suppose that there may be similar signal mechanisms during which TJ proteins can directly act as an early step for the fusion process. However, the detailed cellular events concerning the roles of TJ-associated proteins or TJ itself remain largely uncertain. Additionally, a study has revealed that HCV infection induces reorganization of TJassociated proteins, especially occludin’s dot-like accumulation in the endoplasmic reticulum in association with viral glycoprotein E2 [70]. This association raises the question whether the interaction is required in HCV exocytosis since occludin always transports along the cell membrane to the TJ area. In addition, occludin and claudin-1 are reported to be essential participants in cell–cell transmission utilized by HCV to escape from the host immune response [71]. Liu et al. [72] observed downregulation of expression levels of occludin and claudin-1 during HCV infection in the case of superinfection. Considering the dramatic involvement of occludin and claudin-1 in the HCV entry this step provides a new approach into the evolution of novel therapies and preventive antiviral strategies targeting these TJ proteins. Currently, a human claudin-1-derived peptide named CL58 appears to prohibit HCV entry at a postbinding step [73], offering a potential method for its detection. TJ disruption and polarization defects facilitate viral infection Despite the fact that HCV infection requires the presence of TJ-associated proteins, the role of TJs themselves during viral infection is inconclusive. Several studies have indicated that the disruption of TJ integrity may be induced during viral infection, such as dislocation of claudin-1, occludin and ZO-1 [70]. Wilson et al. [74] revealed a modulation of TJ protein expression, localization and function induced by HCV glycoproteins which were in agreement with previous studies. Mee and colleagues have reported an evident increase in protein expression levels of viral receptors CD81, SR-BI, and claudin-1 upon polarization in Caco-2 cells, with a translocation of claudin-1 between some sites along the cell surfaces [75], indicating the effect of polarization in HCV infection although

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nonpolarized Caco-2 cells remain susceptible for HCVpp entry. Besides, disruption of TJs by depleting calcium level significantly increases HCVpp and HCVcc infections [75]. Considering the fact that HCV gps and particles are preferentially attached to the apical surface of polarized Caco-2 cells, it is suggested here that the disruption of TJs provides an easier access for viral particles to the receptors. Herein a conceptual model is developed where TJs reduced viral access to receptors expressed on the lateral and basolateral cellular surfaces as well as blocked the spread of viruses to new host cells by establishing a physical barrier. Noteworthy, polarization which is largely dependent on functional TJ seems to be greatly related to the receptor expression and subsequent entry though the polarization is not a prerequisite as well as functional TJ. Mee and coworkers showed that HepG2 cells developed a complex of hepatic polarity upon cultivation, which restricted HCV infection with evident effects. This is indicated by the treatment with polarization-stimuli cytokines such as PKA agonists [76]. Other studies have also replenished the important role of activation of PKA involved in HCV infection concerning relocalization of claudin-1 [77]. Since PKA activity triggers cellular signaling pathways and regulates multiple cellular protein properties related to TJ, the distinct mechanism deserves more investigations. In conclusion, the inaccessibility of preventive strategies for HCV infection is still a big challenge with no vaccines available. Therefore, the host receptors and signaling molecules are potential drug targets which may inhibit viral entry and infection. Gastrointestinal infections: EPEC EPEC-secreted effector molecules disrupt TJ Enteropathogenic E. coli (EPEC) is a common pathogenic bacterium of infantile diarrhea in developing countries, characterized as a typically attaching and effacing (A/E) pathogen. Upon initial attachment on the intestinal epithelial cells, EPEC rapidly causes effacement of microvilli of the intestine and recruits polymerized actin to the adherent bacteria in a pedestal-like manner, known as A/E lesions [78]. During the lesion, various bacterial proteins or effector molecules are secreted into host cell membrane generally via an avenue formed by type III secretion system [79]. Currently, at least 21 secreted effector molecules have been recognized, such as Tir, Map, EspF, EspG, EspH, EspI (NleA), NleB-G and EspB, which are classified into LEE effector (encoded on the locus of enterocyte effacement, an pathogenicity island in the genome of EPEC) and non-LEE effector (encoded outside the LEE) due to a different encoding locus (see Fig. 2) [79]. Notably, the

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bacterial outer membrane protein intimin is not delivered into the host but encoded on the LEE as well, which interacts with the translocated intimin receptor (Tir) and triggers close attachment of EPEC to the cell [80]. The intimin-Tir interaction is not only involved in the pedestal formation, but also induces cellular signaling pathways, like tyrosine phosphorylation of host phospholipase, PLCc1 [81]; downregulation of Map-mediated filopodia formation [82]. Map (Mitochondrial-associated protein), one of the LEE effectors, is also involved in TJ disruption, microvilli effacement and mitochondrial dysfunction except filopodia formation [79]. In addition to Tir, other effector molecules are all translocated into the cell and elicit numerous cellular responses via activation of various protein kinases such as protein kinase C (PKC), phospholipase Cc, MLCK and mitogen-activated protein kinases (MAPK), and following downstream effects [78]. Escherichia coli secreted protein F (EspF), which is believed to be central to EPEC pathogenesis, is involved in the clathrin-mediated endocytosis via interactions with SNX9 and N-WASP leading to an actin rearrangement [83]. It is proposed that EspF triggers membrane to cytosol endocytosis of TJ associated proteins as a novel mechanism to disturb barrier functions. Besides, the cooperation of EspF and Map is discovered to cause a maximal level of TER loss compared to each molecule alone, which requires the presence of intimin [84], although whether the binding to Tir is prerequisite or not is still in debate [85]. The ability of Map to disrupt TJ function may result from Map targeting mitochondrial events [79] since changes in ATP concentration can regulate TJ structure as shown above, but currently no convincing evidence has proved it. EspI/NleA (E. coli secreted protein I or non-LEE encoded A), the first-identified non-LEE encoded effector, is a critical factor in the disruption of TJs, for it inhibits COPII-mediated protein secretion and ER to Golgi trafficking [86–88]. As stated above, a lot of cellular events including endocytosis of TJ proteins, membrane trafficking, cellular translocation are involved in the complicated processes of TJ regulation. It’s tempting to speculate that NleA’s virulence to inhibit COPII trafficking may induce TJ disruption via directly blocking traffic of TJ proteins, while the direct action remains to be demonstrated. Tomson et.al suggested that EspG (E. coli secreted protein G) promoted microtubules (MT) disruption in IECs and caused a delay in TER loss accompanied by EspG2/ Orf3 (E. coli secreted protein G or Open reading frames 3, the only EPEC effector so far identified that is not present in EHEC) [89]. One potential explanation is that MT disruption breaks the association with GEF-H1 and thereby inhibits activation of RhoA, which has a greatly regulatory effect for TJ [90]. In addition, disruption of TJ caused by the effector cytotoxic necrotizing factor-1 (CNF-1) is also

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reported to be mediated by Rho protein family activation; and it triggers multiple effects including displacement of TJ-associated proteins, phosphorylation of p-MLC related to F-actin, actin rearrangement and enhanced paracellular permeability consequently [91]. Sason and colleagues indicated that Tir phosphorylated on tyrosine 454 activated PI3K which played a significant role in TJ modulation, suggesting a putative pathway to break down the TJ barrier; in contrast, overexpression of Tir was reported to tighten the barrier proved by an increase in TER caused by EPEC(4tir ? Tir) [92]. To summarize, multiple effectors and molecules secreted by EPEC engage in the disruption of TJs in IECs and a loss of TER, contributing to bacterial virulence. The various effects caused by these effectors are largely mediated by signaling pathways involving a cluster of kinases and phosphatases, such as RhoK, PI3K, though several mechanisms remain obscure. Morphological changes of TJ and potential mechanisms Researchers have demonstrated that EPEC-induced morphological changes of disrupted TJs are characterized by the redistribution of TJ proteins [93]. Furthermore, Michelle and coworkers revealed a decrease in claudin-1occludin and occludin-ZO-1 interactions by co-immunoprecipitation experiments; reorganization of occludin and claudin-1 from TJ areas with a focal loss; as well as ectopic strands arranged along the lateral membrane beneath TJs identified by freeze fracturing [94]. These research results indicate that EPEC disrupts many TJ proteins of the host leading to epithelial barrier dysfunction. However, how EPEC induces the changes in TJ structure and function still remains unknown. Several signaling pathways triggered by distinct effectors have been demonstrated as above. Ivana Simonovic and coworkers indicated that EPEC induced phosphorylation of ezrin, a membrane-cytoskeleton linker and signal transducer, which in turn enhanced ezrin-cytoskeleton association, leading to an abnormal TJ structure [95]. In another analysis, Ivana Simonovic and coworkers proved a reversal in the ratio of phosphorylated to dephosphorylated occludin caused by EPEC infection [95]. Since we have discussed the significant role of phosphorylation of occludin in the TJ assembly and disassembly, it suggests EPEC-induced perturbation of TJ architectures may in part directly result from signaling events correlated with occludin, although the related kinases have not been identified as yet. Notably, the shift of phosphorylated occludin is also observed in Michelle’s experiments [94]. Moreover, previous studies have implicated EPEC infection results in an increase in phosphorylated MLC20 [96]. Other reports have demonstrated that MLC phosphorylation mediated by MLCK

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constricts the perijunctional actomyosin ring [97, 98], thereby affects the TJ-cytoskeleton connection and increases TJ permeability. With these observations, we can draw the conclusion that MLC phosphorylation in part contributes to EPEC-induced increased paracellular permeability by the tension caused by cytoskeleton contraction. Although a number of effector molecules and several possible signaling events have been uncovered, the underlying pathogenesis of diarrhea caused by EPEC remains largely unknown. In the meantime, the exact interactions between TJ and these effectors need more investigations with regard to morphological changes identified in TJ. Gastrointestinal infections: EHEC, EAEC and ETEC Enterohemorrhagic E. coli (EHEC) is an enteric pathogen which leads to wide-range clinical outcomes such as nonbloody diarrhea, hemorrhagic colitis and the hemolyticuremic syndrome [99]. It harbors highly homologous enterophathogenic islands and effectors with Enteropathogenic E. coli, yet shows key different mechanism of action, although the pathogen elicits A/E lesion to the host as well. Howe and coworkers reported that EHEC O157:H7 (strains of serotype) infection in the colon-derived T84 cell caused reduced expression and displacement of ZO-1, occludin, and claudin-2 from TJ, which could be prevented by TGF-b pretreatment via activation of ERK, MAPK and SMAD signaling pathways (see Fig. 3) [100], indicating a protective role for the cytokine TGF-b via up-regulation of claudin-1. However, the effect of claudin-2 alteration remains controversial. Although reduced expression is observed in EHEC-infected T84 cells paralleling to TER loss, addition of claudin-2 into MDCK I cell greatly loosened TJ strands based on claudin-1/4 with TER loss [101]. Potential explanations for this phenomenon are that claudin-induced effects on epithelial barrier are cell-type-specific and claudin may exert a distinct role in specific cell systems; or, simply there remains some hidden signaling pathways which could modulate claudin-2-induced effects. In addition, an in vivo model on C57Bl/6J mice was established to study EHEC infection induced perturbation of TJs, and exploit the mechanism interacted with claudin2 to a further degree. Depending on the mice model, immunofluorescence staining revealed a rearrangement of the TJ proteins occludin and claudin-3 and a more progressive increase of claudin-2 expression. Besides, by qRTPCR alterations of mRNA levels of occludin and claudin-2, claudin-3 have also been observed [102]. We can speculate that the increased claudin-2 expression is attributable to an increase in mRNA transcriptions. The modification of claudin-2 expression has been supposed as a putative

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Fig. 3 Signaling pathways and molecular mechanisms involved in pathogens induced TJ alterations or disruption. H. pylori secrets a virulence factor CagA, phosphorylation of which is mediated by interaction of CagL and integrin a5b1 which in turn induces activation of FAK and Src. Phosphorylated CagA induces SHP-2 phosphatase activation and also binds with Par1b, leading to actin rearrangement and dysfunction of TJ. H. pylori also phosphorylates IL-1RI to activate ROCK. Both ROCK and MLCK induce MLC phosphorylation and lead to TJ disruption, which can be blocked by

PKC. EHEC induces production of TNF, which has a regulatory effect on TJ via PI3K/Akt signaling events. And EHEC-induced TJ disruption can be prevented by TGF-b pretreatment. Adenovirus produces fiber protein, which interacts with CAR, the receptor as well as a transmembrane component of TJ. RSV causes altered expression of TJ proteins via TGF-b1/PKC d/HIF-1 a/NF-jB signaling pathway. HIV-1 infection produces TNF-a, which activates NF-jB and leads to TJ disruption

mechanism to decrease the TER on the colon, since a pathway has been discovered by Mankertz et al. [103]. EHEC induces the production of inflammation cytokine TNF, which can up-regulate the genomic expression of claudin-2 involving PI3K/Akt signaling events [103]. As claudin-2 is required for cation-selective channel formation in the paracellular pathway [104], high level expression probably results in the transformation of a leaky TJ barrier. Furthermore, the increased claudin-2 expression is also observed in active Crohn’s disease and ulcerative colitis [105], both characterized by ‘‘leak-flux diarrhea’’. Compared to EPEC, EHEC exert less profound effects on the TJ function as a barrier due to distinct kinetics while these two pathogens have much in common [106, 107]. EHEC is distinct from EPEC for it can produce Shiga toxin, the most important virulence factor in diarrhea characterized infection. Yet shiga toxin is supposed to translocate from the intestine to the bloodstream [99], which has little relation to non-bloody diarrhea mediated by TJ barrier disruption during early time. Recently another subtype of E. coli, Enteroaggregative E. coli (EAEC) has also been suggested to induce epithelia barrier disruption, similar to EPEC and EHEC. While pathogenesis has not been fully elucidated, the morphological changes in infected cells and relocalization of the TJ proteins occludin and claudin-1 have been observed in response to EAEC infection [108]. Notably, recent studies

have revealed interactions between TJ disruption and enterotoxigenic E. coli (ETEC) infection, another major cause of diarrhea in animals. Ngendahayo et.al demonstrated that ETEC secreted heat-stable toxin b (STb) induced epithelial barrier dysfunction of T84 cell monolayers by redistribution of ZO-1, claudin-1, and occludin and rearrangement of the actin cytoskeleton [109]. It was discussed for the first time, so further studies are required to uncover the signaling pathways involved.

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Gastrointestinal infections: Clostridium perfringens CPE is a pore-forming toxin Clostridium perfringens is the leading cause to type A food poisoning (the most common type of food poisoning caused by the type A entertoxin secreted by C. perfringens) and those of non-foodborne gastrointestinal illnesses, which largely depends on the secreted C. perfringens enterotoxin (CPE). As a single 35 kDa polypeptide [110], CPE is the most significant virulence factor in the pathogenicity of C. perfringens in the human intestine. The molecular mechanism how CPE exerts its toxicity has been studied in several reviews (see Fig. 2) [111–113]. During the infection, CPE firstly accumulates within the bacterium and is delivered into the intestinal lumen upon the lysis of the sporulating cell. Then CPE binds to

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receptors in the intestinal epithelial cells and evokes multiple cellular signal events resulting in cell death and morphological and functional changes on the epithelia. Notably, the C-terminal domain (or C-terminus) of C. perfringens enterotoxin (cCPE) is not cytotoxic but interacts with the second ECL (ECL2) of a group of claudins, whereas the N-terminal is cytotoxic. For instance, claudin-3 (RVP1) [114] and claudin-4 (CPE-R), are initially defined as CPE receptors in early observations [115], and some other members of the claudin family e.g. claudin1, via indirect interactions [112]. A four-step model for CPE action is proposed. After binding, a *90 kDa small complex which is sensitive to SDS (sodium dodecylsulfate) forms and fast oligomerization of the complex constructs a new hexamer of *155 kDa termed CH-1 (CPE hexamer-1) [116]. CH-1 undergoes conformational changes as a prepore complex for insertion into the membrane and causes massive plasma membrane permeability changes in some distinct mammalian cells [117]. In this way CH-1 transforms into a transmembrane pore through which calcium gets easy access to the cytoplasm and leads to cellular apoptosis or oncosis [118]. As the calcium influx increases, the cell permeability is greatly altered and the cellular structure is also damaged. As a result, CPE accesses the exposed basolateral TJ structure and forms a CH-2 complex (*200 kDa) that contains occludin [119, 120], eventually leading to TJ disruption and contributing to CPE-induced cytotoxity. CPE is a claudin modulator Previous studies have suggested that the C-terminal domain (or C-terminus) of CPE selectively removes claudin-4 from TJs, which results in patchy TJs and perturbated barrier function in infected MDCK I cells [121]. A similar discovery has been obtained by Mitchell and coworkers that CPE binding leads claudins away from epithelial membrane [113], suggesting a different way for TJ disruption from the formation of CPE complexes. Besides, CPE has been reported to be somewhat associated with claudin-3, -4, -5, -6, -7, -8, -9 and -14, but whether the interactions contribute to CPE toxicity remained undetermined as yet [113]. Thus, CPE is supposed to be a claudin modulator with interactions with the claudin family. Consistent with its function to bind with claudins, it could be used for reverting claudin-overexpression in tumor therapy and drug transport across physical barriers. For example, overexpression of claudin-3 and/or claudin-4 is common in a variety of tumors, e.g. pancreatic cancer [122], prostate cancer [123], ovarian cancer [124]. Although the role of claudin expression in tumorigenesis remains largely

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obscure, CPE provides an innovative therapy targeting proteins to regulate the tumor development: (1) downregulated expression of the claudins will indirectly affect tumorgenesis; (2) straightforwardly induce tumor cells death by its cytotoxity. Notably, claudin-3 and claudin-4 expressed in the tumor tissue are generally not junctional proteins but mislocalize on the whole cell membrane [125], indicating a more extensive and stronger cytotoxity effect mediated by CPE than at normal circumstances. For example, Kominsky et al. [126] reported that CPE treatment to breast cancer cell lines resulted in rapid and potent cytolysis with a reduction in tumor volume via claudin-3 and claudin-4 interactions. Noteworthy, the non-cytotoxic cCPE is suggested to be applied in chemotherapeutic agents transport by increasing drug delivery to the tumors across the disrupted barrier, revealed by a series of studies [113]. Gastrointestinal infections: Helicobacter pylori CagA and VacA are two major virulence factors Helicobacter pylori infection triggers colonization of gastric epithelial cells and causes various diseases ranging from chronic gastritis, duodenal ulcer to gastric carcinoma in humans. During pathogenesis, H. pylori is believed to cause disruption of cell–cell adhesions and cell polarity, which means to have a profound effect on the epithelial physiological function and integrity. CagA and VacA have been characterized as important virulence factors when bacteria invade the gastric epithelia [127, 136]. CagA is secreted by T4SS pili [128] into the host-cell cytoplasm via interaction of CagL and integrin a5b1 which induces activation of FAK and Src and in turn phosphorylates CagA [129]. The phosphorylation on tyrosine residues leads to multiple cellular effects covering rearrangement of actin cytoskeleton, disruption of apicaljunctional complex, loss of cell polarity [130] and cell elongation [129]. The massive signaling events may induce mild early opening of TJ, which facilitates an interaction of the effector factor and its receptor integrin on the basal side of the cell and, therefore, causes a severe disruption of cell polarity and TJ architecture. As many studies have suggested, the effects of translocated CagA interfering with TJ include recruiting TJ scaffolding proteins ZO-1 and JAM where the bacterium is attached to the cell surface [127, 131] independent of phosphorylation, and forming ZO-1 enriched vesicles both described in MDCK and AGS cells [132]. Krueger’s experiments also revealed an accumulation of p120ctn to perinuclear cytoplasmic vesicles in response to H. pylori infection which can suppress endocytosis of E-cadherin [132]. It is supposed that H. pylori infection may interfere with E-cadherin-mediated cell

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adhesion by p120ctn, however it occurs at adherens junctions (AJs), not TJs. In addition to that, p120ctn has many other regulatory effects on, such as interactions with activity of Rho family GTPases and association of microtubules with the cell junctions [133], which may be involved in H. pylori induced TJ disruption. As CagA induces cellular alterations in these cultured cell lines, several potential signaling pathways which are also important in regulating epithelial functions have been uncovered (see Fig. 3). Hatakeyama reported [134] in a review that CagA activated SHP-2 phosphatase by phosphorylation at tyrosine and forming a physical complex at the plasma membrane and triggered a cluster of downstream effects, including actin rearrangement. More notably, several studies revealed that H. pylori developed strategies to influence the functional TJ complex. Translocated CagA is supposed to bind with Par1b, a regulatory complex that phosphorylates microtubule-associated protein to maintain cell polarity [131, 135]. The combination inhibits the phosphorylation of Par1b and leads to the mislocalization of the complex, contributing to a dysfunction of TJ [127, 135]. In the meantime, VacA by binding with its receptors RPTP a and b is internalized into the cytoplasma via actindependent caveolae-like processes [140], and thereby triggers downstream effects [136, 137]. Although a previous study demonstrated that VacA had no crucial effect on the distribution of TJ-associated proteins like ZO1 and occludin it increased the selective diffusion of ions (Fe3?, Ni2?) and small uncharged molecules, required for microbial growth, via some distinct mechanism rather than specific disruption of TJ [138]. With the effects of early events, in vitro experiments suppose H. pylori could invade the paracellular space and get access to the lamina propria [139]. Its mechanisms have not been discovered as yet. TJ disruption is mediated by ROCK, MLCK, or urease activities Independent of CagA and VacA, H. pylori is demonstrated to disrupt some cell signaling events in association with the regulation of epithelial barrier integrity and function (see Fig. 3). Lapointe and coworkers uncovered that H. pylori activated ROCK through IL-1RI phosphorylation, subsequently leading to a lesser expression and membrane-tocytosol distribution of claudin-4 [140]. Meanwhile, in the epithelial cells exposed to H. pylori SS1, western blotting and immunocytochemistry revealed claudin-4, -5 to a lower expression and also a membrane-to-cytosol distribution of occludin in a MLCK-dependent manner, which increased epithelial permeability [141]. Interestingly, a study has suggested that both ROCK and MLCK activities are associated with MLC phosphorylation

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and mediate the modulation of barrier function of TJ [142]. That study has also demonstrated phosphorylation of MLC and the subsequent result can be attenuated by inactivation of ureB and the encoded urease’s functional activity [142]. With regard to urease activity, Lytton and his colleagues revealed that H. pylori-derived ammonium induced a production of 42 kDa occludin which is derived from N-terminal split of full-length protein in Caco-2 cells, indicating a perturbation of TJ associated protein [143]. Noteworthy, the synergistic effect combined with ammonium and low molecular weight occludin is reversible, which induces additional stress on the integrity of TJ thereby increases the paracellular permeability. Furthermore, Terres et al. [144] observed that the H. pylori-induced TER reduction was blocked by the activation of PKC and the inhibition of PKC activity contributing to the bacterial virulence. Thus these events provide novel signal networks which are independent of translocated bacterial products like CagA and VacA, suggesting the virulence factors are not necessarily crucial for the infection. In the meantime, since signaling pathways involving ROCK, MLCK, PKC are mentioned to be crucial in regulating TJ integrity, it is convincing to suppose that H. pylori generates these cellular events targeting the TJ and modulates the barrier properties of the epithelia to facilitate bacterial infection (see Fig. 3). Respiratory tract infections: Adenovirus CAR is a viral receptor localized in TJ The coxsackievirus and adenovirus receptor (CAR) plays an important role in the infection of coxsackie B viruses and plenty of adenoviruses (except species B [145]) in acting as the virus receptor on the cell surface [146]. Here we put more emphasis on the mechanism of adenoviruses infection, which is a common pathogen seen in human respiratory tracts. Several studies have suggested that generally in polarized cells CAR is localized in epithelial TJs and cell–cell adhesion sites at the basolateral domain in various tissues which is determined by the cytoplasmic tail [147]; CAR is also taken as a transmembrane component of TJs in direct or indirect association with the junctional scaffolding protein ZO-1 [148]. Here it’s demonstrated that CAR functions to mediate cell–cell adhesion via homodimerization. Besides it contributes to barrier function by restricting ion and molecule movements through paracellular pathways in polarized cells [148]. Thus, it is suggested that CAR sequestering to the TJs impedes adenovirus infection because of CAR inaccessibility. Previous studies have indicated that treatments with H2O or EGTA in human ciliated airway epithelia leading to a

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transient break in the barrier or to disruption of TJ facilitates a viral entry by making it accessible to bind with the receptor CAR [149]. Furthermore, Cohen et al. [148] revealed that anti-CAR antiserum effectively inhibited TER decrease induced by a disruption of TJ, and in contrast overexpression of CAR on the basolateral side increased a reduction in TER [150], indicating the role of CAR in barrier function [148]. Following infection, adenovirus is firstly released to the basolateral surface of the epithelial cell to get attachment with its receptor displayed in TJ. However, what signaling proteins and pathways are involved in the disruption of TJ integrity so as the virus can get access to CAR remains to be answered. There is a discovery that may contribute partly to the initial attachment. As revealed by multiple analyses, human airway epithelial tissues express several specific isoforms of hCAR. Unlike most isoforms localizing in adherens areas, a specific isoform hCAREx8 has been identified to localize to the apical surface of epithelia, implying a putative role in initiating the viral infection [151]. Though CAR localizes to the TJ, the nature of its interactions with other TJ-associated proteins remains unclear, except the colocalization with ZO-1 as shown above. Actually, it has been reported that CAR specifically associates with MUPP-1 via its C-terminal PDZ-binding motif in TJ. The absence of CAR by siRNA transfection in TJs is found to evoke reorganization of MUPP-1 to the apicalmost domain rather than TJ areas [152]. MUPP-1 is supposed to be an adaptor for intracellular signaling molecules, consistent with its association with the claudin family [153]. Thereby it raises a novel possibility that adenovirus infection targeting CAR may cause distinct changes of CAR interactions with TJ proteins, by means of signaling events leading to TJ dysfunction and leaky paracellular passages to develop infective stages. Although early studies suggested that cytoplasmic and transmembrane domains within CAR are not required for adenoviral infection [154], it may be just involved in the transient disruption of TJ integrity to the initial attachment of virus fiber to CAR in TJ. Fiber-CAR interaction allows viral entry and escape Fiber, especially the knob domain, is produced from the adenoviral capsid which predominantly binds to viral receptors. During entry, virus undergoes binding with receptors, uncoating and endocytosis process. Burckhardt and coworkers demonstrated that the viral fiber-CAR interactions together with coreceptor integrin binding successively triggered diffusive motions, actomyosin-2dependent drifts and confined motions (for integrin) [155]. Then these motions elicited separation of fiber from the

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virus and disclosure of membrane-lytic internal capsid protein VI, which eventually led to viral endocytosis in a dynamin and actin-dependent manner, as well as viral translocation from endosomes to the cytosol, as the experiments presented [155]. In addition to viral entry, CAR-fiber interaction is also described to be involved in viral escape and spread processes. As many articles demonstrated, adenoviruses utilized fiber protein to interact with CAR, which competed with CAR–CAR self-interaction in cell–cell adhesion [150]. In this way, fiber-CAR interaction induced TER reduction and the virus spread through the paracellular passage to a new host cell or to the lung, accounting for the observation that infected epithelial cells produced excess fiber proteins [150]. There were reports of early attempts to treat certain diseases by exploiting adenovirus vectors to deliver specific genes to human airway epithelium but it didn’t turn out well [156]. This explains why the replication-defective adenovirus vectors do not have the capacity to produce excess fibers to disrupt CAR–CAR interactions and thereby move to targeted tissues. Thus, fiber-CAR interaction apparently evolves to serve two equally important, yet fundamentally different steps in the adenovirus life cycle, including receptor-mediated entry and viral escape. In addition to CAR, other cellular molecules and proteins like desmoglein (DSG) and CD46 have also been identified as the receptors for adenovirus species B, such as Ad-3. Notably, while adenovirus serotype 3 utilized DSG-2 as the receptor rather than CAR, it still displayed the ability to trigger a transient opening of intercellular junctions dependent on the multimerization of the fiber knob domains. Moreover, the attachment to the cell could be blocked by soluble fiber protein in CAR and CD46 binding serotypes while the Ad3-DSG interaction is ineffectively blocked [145]. Generally speaking, respiratory tract infective viruses must traverse respiratory epithelia in the course of infection, but as the previous studies have suggested, adenovirus initially interacts with receptor CAR which is somewhat unreachable from the epithelial surface. We can speculate that infection by adenovirus in vivo requires the loss of epithelial integrity including TJ disruption or associations with alternative receptors located on the apical side. TJ integrity and function can be modulated by various signaling events and molecules as shown in the TJ assembly regulation. Thus much more investigations are required to figure out any possible modulatory mechanism present in TJ during adenoviral infection and the possibility of the presence of other available receptors. As an aside, adenovirus can cause failure in TJ formation and polarity defects in epithelia cells by the oncoprotein E4-ORF1 protein, which leads to mislocalization and inactivation of PDZ

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proteins which have crucial regulatory effects in TJ [157] which are consistent with the development of cancer related to the virus. Respiratory tract infections: RSV Respiratory syncytial virus (RSV) is a significant respiratory virus that mainly leads to serious pediatric respiratory tract disease in infants and young adults. It principally invades upper respiratory tract cells, especially nasal epithelial cells where the TJs are suggested to play a crucial role as the first line of defense. In establishing an RSV infection model using human telomerase reverse transcriptase transfected HNECs, an increase in expression of claudin-4 is demonstrated which is mediated via a TGF-b1/PKC d/HIF-1 a/NF-jB signaling pathway (see Fig. 3) [158]. Together, an increase in expression of occludin and ZO-1 are also shown by immunocytochemistry at 24 h after RSV infection [158]. However, claudin-4 and occludin are proven not to be receptors of RSV, but possibly contribute to budding of RSV while the mechanism remains largely unknown. Besides, gene chip analysis revealed up-regulation of TJ components claudin-2, -4, -7, -9, -14, and -19, occludin, ZO-2, in HNECs infected with RSV [158]. Furthermore, Tsutsumi [159] proved that RSV infected the nasal epithelial cells only from the apical side.

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HIV-1 is supposed to decrease the transepithelial resistance across the columnar epithelial cells directly by perturbation of TJ. Nazli and coworkers demonstrated that following HIV-1 exposure, a disruption of TJ occurred to genital tract epithelial cells due to a down-regulation of TJ protein expression and mRNA level including claudin-2, occludin and ZO-1, dependent of HIV envelope glycoprotein gp120 [164]. This study and other research also indicated that exposure to HIV induced the massive production of inflammatory cytokines in the genital monolayer, especially containing TNF-a (see Fig. 3) [165]. TNF-a has been reported to induce a significant downregulation of TJ proteins and cause the impairment of TJ barrier function and leakage mediated by NF-jB activation [166]. Thus HIV-1-induced increased permeability in genital epithelia cells allows a small amount but significant viral and bacterial translocation across the epithelia to the inside of the body. Besides, recent studies demonstrated epithelial cell infection by HIV can be gp120-independent. Experiments described that claudin-7 expression on the surface of CD4(-) cells made them much more vulnerable to HIV-1 attack [167]. Thus, claudin-7 is suggested to be a possible receptor or a viral envelope ligand for HIV-1 infection in CD4(-) cells. Considering the multiple expression of claudin-7 in the urogenital system, whether claudin-7 on the genital epithelial cells promotes HIV infection requires to be answered. Pharmacological prospects

Female reproductive tract infections: sexually transmitted pathogens There is emerging evidence indicating that female reproductive tract is fairly susceptile to sexually transmitted infections (STIs) and the epithelial lining participates as the primary barrier. The upper reproductive tract is covered by a continuous monolayer of columnar epithelial cells characterized by TJ structures which are responsible for the maintenance of the physical and functional barrier to destructive pathogens. STI pathogens such as HIV, HSV (herpes simplex virus), and Chlamydia, universally need to traverse or disrupt the mucosal epithelium to initiate infection. However, instead of TJ, HSV [160] and Chlamydia [161] are reported to associate with nectin-1, an important adhesion protein, leading to disruption of AJs although Chlamydia trachomatis infection of fallopian tubes also disrupt cell polarity and claudin-1-mediated cell adhesion [162]; Neisseria gonorrhoeae infection induces redistribution of E-cadherin at AJ as well as its adapter protein b-catenin [163]. Here we highlight a viral STI-HIV, which has been demonstrated to casue evident alteration in the morphology and function of TJs as a tool to produce infection in the genital tract.

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In recent years, complicated interactions of epithelial TJs and pathogen infections are unraveled with considerable efforts. Based on these crucial discoveries, preventive and therapeutic approaches are greatly widened by exploring new drugs or agents targeting viral/bacterial life cycles and host cell factors. For instance, the HCV entry into epithelial cells can be targeted prospectively for pharmacological strategies since viral glycoprotein interactions with host cell factors have been largely elucidated. Promising drug designs may interfere with HCV infection on distinct stages including viral attachment and binding with receptors, postbinding events, internalization and fusion, and even the viral escape process. So far several potential approaches have been made for example with the development of anti-claudin-1 antibodies [61]. These may be effective in the treatment of other pathogen infections and clinical diseases as well, by neutralizing antibodies or siRNAs against these involved effectors and molecules [168]. In addition, interferoninduced transmembrane protein 1 (IFITM1) is demonstrated to interact with HCV coreceptors, including CD81 and occludin, to suppress viral entry, indicating a promising method to promote type I IFN treatment by enhancing

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IFITM1 expression [169]. Besides, a potential therapy for prostate cancer is suggested to target claudin-4 by the utilization of CPE, which can also be exploited in other tumors stated above, although the side effects of CPE need to be carefully estimated before application in clinical practice [125]. In addition to therapeutic interventions via the TJ to interfere lifecycle of these closely related pathogen infections, the fundamental functions maintained by TJ integrity should also be taken into pharmacological considerations. As TJs are the dominant factors of paracellular permeability, they may largely define the paracellular transport of drugs or agents if favorably adapted or modified. As discussed above, the TJ component claudin family primarily determines the barrier properties and specific claudin may have different effects on the barrier. For example, claudin2 forms a specific passage for small cations and water [104] while claudin-17 offers a channel for anion [170]. Thus claudins are potential targets for distinct permeability of certain drugs and agents as modulation of claudins may cause a transient and reversible opening of TJ. In an experiment with Caco-2/HT29-MTX co-cultured cells goblet cell-specific trimethyl chitosan nanoparticles were uptaken and translocated via opening of the epithelial TJs mediated by decreased expression of claudin-4 [171]. Actually, recent advances in nanoparticles have raised more possibilities of applying them to drug delivery through TJs, which can cause TJs opening transiently while the adverse effects have to be clarified [172]. Currently, several potential mechanisms have been summarized in a review [173] with the ability to transiently open TJ without any true lesion to TJ integrity, such as Ca2? chelation, C. perfringens enterotoxin (CPE), chitosan and fatty acids. Once these novel approaches are applied in clinical therapy, it will immensely facilitate drug absorption and promote tissue-targeted drug transportation efficiency by overcoming the TJ barrier.

Concluding remarks TJs are dynamic structures that block up the paracellular space between adjacent epithelial cells and function as barrier and fence. In this review TJs are demonstrated to be of great importance involved in a multitude of infections. This is shown in two aspects: (1) TJ proteins can either act as receptors for pathogen entry or interact with viral/bacterial effector molecules as an essential step for attachment, binding, internalization or fusion processes; (2) TJ are altered in a morphological, structural or functional pattern to disrupt paracellular permeability to facilitate pathogen entry and spread. However, much more investigations are in great demand since detailed mechanisms and signaling

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pathways are largely undiscovered as yet. Considering these intricate interactions, application prospects of therapeutic and pharmacological approaches targeting TJs should be highlighted. Acknowledgments We are indebted to all members of the Sperm Laboratory at Zhejiang University for their enlightening discussion. Dr. Hans-U. Dahms is thanked for the critical reading of an earlier draft of this MS. This project was supported in part the National Natural Science Foundation of China (No. 81100393 and 41276151). Conflict of interest Authors declare there is no conflict of interest regarding this work.

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