Molecular mechanisms of Shigella effector proteins: a common pathogen among diarrheic pediatric population

The first barrier to microbial infection is mucin glycoprotein. Shigella can glycosylate and remodel the mucus barrier to its benefit. Mucin is categorized into three types, including cell surface mucin, non-oligomeric gel-forming mucin, and oligomeric gel-forming mucus [23]. Gel-forming mucin as a major component of mucus can be induced by proinflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), neutrophil elastase, and microbial product [24, 25]. Stimulation of a specific gel-forming mucin namely Mu5Ac by Shigella flexneri (S. flexneri) leads to the accumulation of a gel-like structure on the apical surface that facilitates access to S. flexneri, thus leading to invasion. However, S. flexneri attempts to reduce the secretion of gel-forming mucus to reduce its distance from the epithelial cell. Nevertheless, S. flexneri can modify the glycosylation of mucin, resulting in changing the structure to its benefit. This phenomenon is T3SS dependent, and the MxiD mutant that lacks T3SS efficiency does not exhibit this phenomenon [26]. Given that Shigella cannot invade the apical epithelial surface, the target M cells penetrate the epithelial barrier. M cells are particular cells in mucosal-associated lymphoid tissue (MALT) that play a significant role in the transport of antigen from lumen to antigen-presenting cells (APC). M cells with their thin microvilli along with the absence of surface glycoprotein are exposed to the Shigella invasion [27]. The entrance of S. flexneri to the M cells leads to the recurrence of polymorphonuclear neutrophils (PMN) and increase in the size of M cell. S. flexneri cannot invade the apical membrane of the colonic cell; however, the recurrence of PMN leads to the destruction of epithelial conjunction and helps Shigella reach the basolateral space [28, 29]. Inflammation, which is induced by PMN, leads to greater permeability and further passage of S. flexneri. Thus, S. flexneri can pass through the epithelial cell by M cells or directly through permeability induced by PMN. After this, the bacteria swallowed by dendritic cells (DCs) and macrophages in the pocket of M cells degrade; however, S. flexneri can induce apoptosis in this APC and release itself [30]. In the first step, S. flexneri ruptures the vacuole and escapes to the cytoplasm; then, it fully duplicates in the cytoplasm. In the final step, Shigella induces host cell apoptosis to make itself free. Mucosal inflammation is partly induced by peptidoglycan and sensed by the nucleotide-binding oligomerization domain-1 (NOD1). Being a cytosolic pattern recognition molecule, NOD1 can bind with the peptidoglycan in the cell wall structure and mediate cascade signaling, leading to the stimulation of inflammatory responses. NOD1 specifically recognizes gamma-D-glutamyl-meso-diaminopimelic acid in the peptidoglycan of gram-negative bacteria.

After sensing this ligand by NOD1, its adaptor receptor-interacting serine/threonine-protein kinase 2 (RIP2) recurs and is finally phosphorylated [31]. This phosphorylation leads to the activation of the tumor growth factor-β (TGF-β) activating kinase 1 (TAK1). This cascade leads to the activation IKK complex and is, finally, phosphorylated and causes proteasomal degradation for the NF-κB inhibitor. This effect leads to the release of p50 and p65 subunits of NF-κB, thus producing the antimicrobial peptide, chemokines, and cytokines [32].

Finally, this sensing leads to the activation of nuclear factor kappa-B (NF-κB); in turn, NF-κB leads to transcription of interleukin 8 (IL-8), which is a major factor in the recurrence of neutrophil [33]. NOD1 also leads to the upregulated expression of intracellular adhesion molecule-1 (ICAM-1) through NF-κB signaling. ICAM as transmembrane glycoprotein has a receptor role in the β2 integrin of leukocytes. This upregulation leads to greater recurrence of neutrophils to the infection site [34].

Intestinal epithelium plays a protective role in the luminal barrier and inhibits the penetration of pathogen and nonpathogenic bacteria. However, the basolateral surface is a clean area that does not encounter lipopolysaccharides (LPS). Thus, presentation of S. flexneri in the basolateral membrane may stimulate an epithelial response [35]. LPS is a major factor in forming an interaction with epithelial cells in the basolateral position, and S. flexneri with defects in LPS structure may affect the ability of bacteria to mediate the basolateral attachment. Attachment and recognition of LPS lead to the activation of extracellular signal-regulated kinase (ERK) and the initiation of inflammatory responses. The inflammatory response, in turn, results in PMN recurrence [36]. Invasion of apical epithelial cells is limited, which is proportional to the basolateral surface. Moreover, the addition of M cells to the apical surface mediates increased invasion by S. flexneri [29]. LPS and intermediate metabolite activate the inflammatory response, and tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) protein interacting with the forkhead-associated domain (TIFA) may induce the activation of TRAF2 and TRAF6, leading to activation of the inhibitor of the nuclear factor-kB kinase (IKK). β-heptose 1,7-biphosphate as an intermediate metabolite of the LPS biosynthesis pathway mediates the activation and oligomerization of TIFA. TIFA activation prompts proinflammatory gene expression. Interestingly, β-heptose 1,7-bisphosphate causes a delay in TIFA activation and should be processed intracellularly to induce inflammation. As a metabolite, ADP heptose can mediate rapid TIFA activation and recognize new pathogen-associated molecular patterns (PAMPs) [37]. Although NOD-1 can detect S. flexneri invasion, NOD-1 facilitates detecting the early phase of the invasion. However, TIFA performs the next or final recognition phase for Shigella invasion through bacterial replication sensing [38]. On the other hand, the O-antigen of LPS interacts with gangliosides on the T-cell surface to mediate injection of the T3SS effector; this injection needs actin polymerization [39]. Shigella can only inject effector protein inside the lymphocyte without invasion, a phenomenon that used to be called injection-only. Many immune cells can be affected by injection-only such as B cell, T CD4+, T CD8+, and memory B cells [40]. Although TLR4-MD2-CD14 mediates LPS recognition on the cell surface of the eukaryotic cell, cytosolic plant disease resistance-like protein (CARD4/NOD1) mediates cytosolic recognition of the LPS. Activation and oligomerization of CARD4 by LPS of invasive Shigella sufficiently induce NF-κB and c-Jun N-terminal kinases (JNKs) activation [35]. This activation involves not only the JNKs kinase activity but also the c-Jun phosphorylation, which finally leads to the regulation of inflammatory response [41]. LPS of Shigella is hexa-acylated and can be modified when the growth of bacteria inside the epithelial cell occurs. This modification made by hypoacetylation provides a chance for inducing an immune response, reducing the capacity to induce oxidative burst by the PMN, and downregulating the release of IL-1β from the infected macrophage [42]. Altogether, the response released by Shigella facilitates survival, in all probability.

After the multiplication of Shigella in a host cell, damage and inflammation occur. One strategy to terminate intracellular bacterial life is to program cell death. Recognition of components such as LPS and T3SS via nod-like receptor (NLR) and TLRs leads to inflammatory caspase activation [126]. Furthermore, the LPS recognition triggers caspase-4 and caspase-11 to activate pyroptosis, resulting in cell death and intestinal epithelial cell shedding [127]. Pyroptosis is activated through NLR and mediated by IL-18 and IL-1β, resulting in membrane rupture. Membrane rupture in the case of pyroptosis cells by caspase-1 leads to ion venting and inflammatory response [128]. The lipid-A component of cytoplasmic LPS can directly bind with the caspase-11, resulting in the activation of the inflammasome and pyroptosis [129]. However, Shigella can prevent epithelial cell death before total duplication [130]. Cell death also is induced through mitochondrial injury regulated through the interaction between cyclophilin D and Bcl-2/19kDa protein 3 (Bnip3). NOD1 detects bacterial components and causes protection against the activation of Bnip3 and cyclophilin D, ensuring protection against epithelial cell death. Therefore, NOD1 may mediate protection against cell death in nonmyeloid cells [131]. Protection against cell death by NOD1 depends on the ability of NOD1 to induce NF-κB [132]. After the entrance of Shigella to the epithelial cell, membrane ruffle forms around the bacteria and leads to the recurrence of NOD1 and the component of NOD1 downstream signaling NF-κB essential modulator (NEMO) to the bacterial positions. Localization of NOD1 in the plasma membrane depends on F-actin [133]. Another type of NOD1, NLRs as an immune sensor for bacterial components, consist of two parts: nucleotide-binding domain (NBD) and leucine-rich repeat (LRR). Being autoinhibitory, LRR inhibits the activation of the NBD domain and, as a sensor, directly or indirectly detects the microbial components [134, 135]. In macrophages, the activation of NLRC4 (nod-like receptor C4) and NLRP3 (nod-like receptor P3) commenced, thus inducing pyroptosis and secretion of IL-1β and IL-18 [136]. MxiH needle protein of T3SS is detected by neuronal apoptosis inhibitory protein (NAIP), leading to the activation of NLRC4 inflammasome [137]. This activation mediates the release and activation of human neuronal apoptosis inhibitory protein (hNAIP). hNAIP can sense T3SS components and flagellin and cause NLRC4 inflammasome activation. However, given that S. flexneri does not express flagellin, MxiH has a significant role in the activation of NLRC4 [138]. MxiH is also injected into the host cytoplasm and modulates antimicrobial gene transcription [139]. NLRC4 can detect conserved T3SS components and distinguish between T3SS-positive and T3SS-negative bacteria [136]. However, recognition of and response to MxiH can be done in a dose-dependent manner (in low doses), thus leading to the activation of caspase and pyroptosis. However, in high doses, activated NLRP3 leads to pyronecrosis [138]. Pyronecrosis is considered as a subtype of necrosis and is a caspase-independent cell death pathway. Pyronecrosis is activated through the NAIP-dependent pathway, and this activation is induced by the mutation of the NAIP gene or microbial pathogens. Therefore, altogether, Shigella may trigger cell death through both apoptosis and necrosis [140]. It should be noticed that almost in early events, the caspase-1-dependent mechanism mediates apoptosis; however, in later events, caspase-1-independent apoptosis occurs by lipid A [141]. This event explains that at the initial phase of the infection, whose bacterial dose is low, caspase-dependent death occurs; however, after the replication, caspase-independent apoptosis occurs with a considerable amount of lipid A. NAIP2 as an NLR family can directly bind with the T3SS rod proteins and induce caspase-1 activation. Upon binding with its ligand, NAIPs with NLRC4 formed an inflammasome. NAIP2, as an immune sensor, regulates the oligomerization of NLRC4 and the formation of the NAIP2-NLRC4 complex [142]. As a homolog to the NAIP1 in the mouse model, hNAIP can recognize the T3SS needle MxiH and activate NLRC4 inflammasome [138, 143]. Interestingly, in the intracellular life of Shigella, the T3SS is dampened but reactivated during actin-based motility and cell-cell spread [144] (see “IpaH family” section).

Shiga toxin, a part of A-B toxin, is a pentamer B subunit that functions by connecting toxin to the host cell. Subunit A penetrates the host cell after connection with subunit B. Subunit B connects to the neutral glycolipid globotriaosylceramide (Gb3), and it is endocytosed. After endocytosis, it is transported to the Golgi and, conversely, is translocated to the ER. In the ER, subunit A enzymatically activates and transfers it to the cytosol to remove adenine from 28S RNA, which leads to inhibition of protein synthesis [199]. In the subunit A, the region between amino acids 248–251 mediates trypsin sensitivity and cleavages subunit A into A1 and A2 [200]. Gb3 as a significant receptor for toxin and cell line deficiency in Gb3 is insensitive to the toxin. In addition, Fabry’s disease with overexpressed Gb3 attenuates sensitivity to the Shiga toxin, perhaps due to the spread of toxin in the whole body instead of one position [201]. Shiga toxin also mediates Ca+ influx and ATP release from infected HeLa cells. ATP release mediates ATP signaling through purinergic receptor P2X, which in turn leads to Ca+ influx and cellular damage. Finally, it produces Shiga toxin containing microvesicle [202]. Shiga toxin mediates unfolded protein response mediated by ER and yields apoptosis for epithelial, lymphoid, and endothelial cells [203]. The toxin can be translocated and shed by stimulated blood cells. Translocation of toxin finally leads to its release by microvesicle and its attachment to a target cell such as a renal cell [204]. A human endothelial cell may have different amounts of Gb3 on the cell surface, and exposure to the LPS increases the amount of Gb3 sixfold. Interestingly, the level of Gb3 in renal cells is much more than the endothelial cell and may explain the sensitivity of the renal cell to the toxin [205] (Fig. 3).

In general, interference with the innate and adaptive immune responses in children during primary infection leads to the high susceptibility of children to shigellosis than adults [206]. The difference in human susceptibility to Shigella infection is rooted in the discrepancies between key components of the human innate defense barrier present in the colon [139, 206]. T3SS is the main weapon of Shigella to dampen host defenses. Shigella T3SS effectors target key cellular pathways of gut resident macrophages and enterocytes and modulate the important host cell functions [16]. Shigella T3SS effectors affect different signaling pathways involved in trafficking, cell viability, host cell actin cytoskeleton dynamics, and NF-κB-mediated inflammatory pathways [42]. On the other hand, Shigella leads to the reprogramming of gene expression in infected enterocytes and contributes to the downregulation of CCL20 production. CCL20 is a chemokine mediating DCs recruitment [207].

One mechanism for blocking the spread of cell-to-cell Shigella is GTP-binding protein (Septin). Septin has a role in cell division, but this protein can also activate through actin tail produced via cytoplasmic bacteria. TNF-α may stimulate the formation of septin and surround cytoplasmic bacteria that form the actin tail [208]. In other words, septins make a cage-like structure to contain the free movement of Shigella. Septin can sense micron-scale membrane curvature and bring the proteins back to the bacterial position. Cardiolipin with anionic properties has the presence on the bacterial division site. Cardiolipin via curvature property may trigger activation and migration of septin to the bacterial location [209]. Septin recruits to the IcsA position that mediates actin polymerization. Adaptor p62 protein also recurs to the septin location and mediates autophagy [210]. Cardiolipin is present in both the inner and outer membranes of the cell. PbgA as a transporter takes cardiolipin from the inner to outer cell membrane. The mutant of pbgA appears to be devoid of cardiolipin in the outer membrane cell. pgbA mutation cannot keep IcsA properly fixed in the outer membrane. Altogether, cardiolipin in the inner membrane is responsible for cell division and in the outer membrane responsible for properly localized IcsA [211]. IgG against IcsA and IpaB is shown to be protective, and it reduces the severity of S. flexneri infection [212]. Paneth cells secrete antimicrobial peptides such as LL-37 and defensin. Interestingly, S. flexneri can downregulate the secretion of this antimicrobial peptide. MxiE, as a bacterial regulator, can also regulate innate immune responses and suppress the expression of the antimicrobial peptide by intestinal cells. S. flexneri mediates suppression of chemokine CCL20 [139, 206].

Neutrophil destroys Shigella intracellularly or extracellularly; in the intracellular mode, it occurs following the engulfment of Shigella into vacuole present in the lysosome and is digested [213]. In the extracellular mode, Shigella is trapped and killed using neutrophil extracellular trap (NET). A critical component of NET is elastase that mediates the destruction of both outer membrane protein and virulence factor of Shigella in a lower concentration proportional to other bacterial proteins [214, 215]. After the interaction of LPS-TLR4, pentraxin 3 production is stimulated. Pentraxin 3 as a long pentraxin together with a short pentraxin such as C-reactive protein (CRP) forms acute-phase proteins. Pentraxin can bind with Shigella and interfere with Shigella epithelial invasion [216]. After the entrance of S. flexneri inside the macrophage, 34 KD outer membrane protein (OMP) can be detected by TLR-2. OMP induces expression of TRAF6 and MyD88 and facilitates the phosphorylation and activation of p38. It can also stimulate macrophages to produce chemokine and cytokine and upregulate the expression of MHC-II. All of these features are dependent on the expression of TLR-2 on the macrophage surface [217]. The 34 KD OMP exposed on the surface and antigenically conserved in S. flexneri mediates the induction of proinflammatory cytokines in macrophage, and it plays a protective role in stimulating an immune response [218]. In primary infection due to missing T-cell response, ultimately, eradication of Shigella failed; however, in the case of secondary infection, Th17 produces IL-17A which yields restricted bacterial infection.

Interestingly, Th17 induction shows a stronger response over Th1 [219]. Although the adaptive immune response has a significant role in controlling intracellular infection, CD8+ cell as an adaptive immune cell has minor protection against S. flexneri infection. Interestingly, T-cell response can be attenuated when APC is infected with S. flexneri [220]. Although many vaccines have been proposed so far, only vaccines against O-polysaccharide were developed, and they ensure almost 2-year protection. This vaccine has functional capabilities and mediates serum bactericidal activities [221, 222]. However, this protectivity by IgG is serogroup specific against shigellosis. The presence of specific IgG and IgA leads to a protective effect against homologous Shigella species [222‐224] (Fig. 4). S. flexneri can induce hyperinflammatory response through EGFR and NOD2. Shigella is inducing indoleamine 2,3-dioxygenases 1 (IDO1) by EGFR and NOD2. The above measure and the host cell response mediate immune hemostasis and disrupt IDO1 production through EGFR abrogation, and NOD2 signaling leads to imbalanced response as well as disrupted colon epithelial barrier and cytokine response [225]. Thus, S. flexneri induces IDO1 to mediate immune hemostasis, modulate cytokine secretion, and reduce cytokine consequences.

Horizontal gene transfer (HGT) is the lateral exchange of genes between organisms and has been revealed for various organisms such as bacteria and viruses [226]. In total, the three main mechanisms of HGT are transformation, transduction, and conjugation. Conjugation is the most studied HGT mechanism in the human intestine, and it requires cell-to-cell contact [227]. Plasmids and the mobile genetic elements can be transferred through conjugative machinery. Bacteria in the gut environment have suitable conditions such as stable temperature, sufficient and permanent food resources, fixed physiological conditions, a large number of phages and bacterial cells, and plenty of opportunities for horizontal gene exchange [228]. It is revealed that the frequency of HGT in infants’ meconium and early fecal samples is higher than that in adults [229]. Genes that are widely transferred among bacterial genera and species encode proteins involved in fitness and multiple cycle-like alterations of gene expression. Prokaryotes of intestinal microbiome are reservoir of closely related antimicrobial resistance genes [228]. Antibiotic resistance genes spread between bacteria in gut environments through HGT and expression of resistance genes from other strains. Notably, HGTs along with the bacteriophages, conjugative transposons, plasmids, and integrons have the main role in the transfer of genes and acquisition of pathogenicity by pathogenic human enteric pathobionts, and it leads to the expansion of virulence traits and antibiotic resistance [228].