Host-Pathogen Interactions in Pathophysiology of Diarrheal Disorders

64.1 Introduction

Gastric acid is one of the most important host defenses against infectious agents that enter the gastrointestinal tract (GIT). At pH < 3, gastric juice can kill bacteria within 15 min and hypochlorhydria (caused by drugs that inhibit acid secretion, atrophic gastritis, or gastric surgery) increases the risk of enteric infections. GI motility also provides host defense by reducing the adherence of pathogenic microbes to the GI mucosa. In addition to gastric acid and GI motility, there is a complex, multifaceted barrier that protects the underlying mucosa from noxious agents that enter the GI tract as well as provides a healthy separation between the intestinal microbiota and intestinal epithelial cells. This barrier includes a robust mucus layer secreted by both goblet cells and intestinal epithelial cells, antimicrobial peptides produced primarily by Paneth cells but also intestinal epithelial cells, IgA, the intestinal epithelial cells themselves, and intercellular tight junctions that protect the paracellular space. Further, the gut microbiota consisting of ~ 10 ¹⁴ resident bacteria promotes resistance to infection by competing with pathogens for resources and limiting pathogenic bacterial colonization. Besides the presence of these protective mechanisms in the host, microbial pathogens have developed elaborate machinery to subvert host functions and cause disease. For instance, many pathogens harness the cytoskeleton to mediate their own uptake into host cells or utilize vesicular trafficking pathways to avoid antigen presentation and their delivery into harmful intracellular compartments. In the course of ensuring their own survival and transmission to new hosts, these pathogens often produce a series of undesirable outcomes. Of primary concern in the GIT is diarrhea with an estimated mortality rate of one million deaths in children < 5 years, especially in developing nations. Traditional diarrheal pathogens have also been associated with chronic disease in both developing and modernized countries. For example, repeated infections before age 2 with enteric pathogens are associated with a permanent reduction in both height and IQ and impact lifelong earning potential. Also, up to one-third of patients develop prolonged GI complaints following acute bacterial gastroenteritis and a subset of those acquire postinfectious irritable bowel syndrome (IBS). Thus, even transient infection with enteric pathogens may have profound and life-altering consequences requiring increased understanding of host-pathogen interactions. Diarrhea, which is characterized by an increase in water content, volume, or frequency of stools, generally occurs when there is either a decrease in fluid/electrolyte absorption or increase in secretion of water and electrolytes in response to an infection. The major epithelial processes contributing to pathophysiology of diarrhea include (i) perturbation of barrier function; (ii) a decrease in the function of Na ⁺ absorbing Na ⁺ /H ⁺ exchanger family isoform 3 and the Cl ⁻ absorbing SLC26A3 ( ${Cl}^{-} / {HCO}_{3}^{-}$ ${Cl}^{-} / {HCO}_{3}^{-}$ exchanger, DRA), and/or (iii) activation of the chloride-secreting channel, the CFTR. The following sections address the pathophysiology of the common diarrheogenic pathogens that provide insight into potential host-based therapeutic interventions by increasing our understanding of pathogen-specific virulence factors and signaling cascades affecting a wide-range of host-cellular functions to elicit luminal fluid accumulation causing diarrhea.

This chapter will highlight host epithelial-microbial interactions of various diarrheogenic pathogens that are classified as below:

I.
Enterotoxin-producing organisms: Vibrio cholerae and enterotoxigenic Escherichia coli or viruses such as Rotavirus.
II.
Invasive organisms: Salmonella spp., Shigella spp., Campylobacter spp., Cryptosporidium parvum .
III.
Inflammatory diarrhea: Cytotoxin-producing noninvasive bacteria, for example, Clostridium difficile , enterohemorrhagic E. coli .
IV.
Other pathogens: Nontoxigenic enteropathogenic E. coli and Helicobacter pylori .

64.2 Enterotoxin-Producing Organisms

Enterotoxins affect host target cells in a robust and rapid way. The following sections will discuss host-microbial interactions of microorganisms that are adherent as well as toxigenic such as V. cholerae , some E. coli strains and rotaviruses. The major receptors of the toxins and mechanisms of their actions are summarized in Table 64.1 .

Table 64.1

Enterotoxins Causing Watery Diarrhea: Host Receptor and Mode of Action

Toxin	Organism	Host Receptor	Mode of Action
Cholera toxin (CT)	V. cholerae	G _M1 ganglioside	↑ cAMP; ↑ CFTR; ↓ NHE3
Heat stable toxin (ST _a )	Enterotoxigenic E. coli (ETEC)	Gunaylate cyclase (GCC)	↑ cGMP; ↑ cAMP; ↑ CFTR; ↓ NHE3
Heat-labile toxin LT I	ETEC	G _M1 ganglioside	↑ cAMP
LT IIa LTIIB	ETEC	G _D1a ganglioside G _D1b ganglioside	↑ cAMP ↑ cAMP
NSP4	Rotavirus	Integrins TLR2	↑ Ca ^{2 +} ↑ TMEM16A; ↓ SGLT1 ↑ Serotonin; ↑ VIP

64.2.1 V. cholerae

V. cholerae is the causative agent of cholera. Cholera is perhaps the most dramatic of the diarrheal diseases causing copious amounts of fluid production, primarily due to the presence of cholera toxin (CT). Extensive research over the past decades has resulted in CT being one of the best understood bacterial toxins.

64.2.1.1 Structure of Cholera Toxin

While V. cholerae secretes a number of toxins, the most widely recognized and perhaps the most significant in terms of disease is CT. Characterization of CT began in the 1960s. Initial experiments involved bacterial lysates or filtered culture supernatants of V. cholerae that were found to inhibit sodium uptake. During attempts to purify the active toxin, the authors inadvertently discovered the two-component nature of CT. Initial preparation and dialysis allowed for retention of one component in the dialysis bag and another component in the surrounding media. Neither component was functional alone but when combined the putative CT was reformed. In later studies, the same group utilized antibody against CT to identify the components of the toxin at a more purified level. They identified what was referred to as choleragen, a single active unit, and choleragenoid, an inactive five-component protein that also reacted to the antibody. Through further studies, including the ultimate crystallization of the toxin, it became clear that CT contains a single A subunit consisting of two peptides CTA ₁ and CTA ₂ (22 and 5 kDa, respectively) joined by a disulfide bond located centrally and 5 B subunits (CTB-11 kDa) arranged in a ring-like structure peripherally. The choleragen, previously described by Finkelstein et al., represents the intact CT while the inactive choleragenoid consists of the pentameric B subunit ring. The CTA subunit is itself the active portion of the molecule, specifically the CTA ₁ peptide causing the disease phenotype. The CTB subunit binds to the host-epithelial receptor G _M1 and in subsequent retrograde transmission through the Golgi, delivers CTA to target cells (discussed below). Currently, recombinant CTB (rCTB) is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that neutralizes CT in the gut. Interestingly, recent studies have revealed antiinflammatory effects of CTB in vivo; CTB was shown to mitigate intestinal inflammation associated with Crohn’s disease in mice and humans.

64.2.1.2 Mode of Action of CT

64.2.1.2.1 Interaction of CT With Receptor and Entry Into Host Cytosol

64.2.1.2.1.1

CT Receptor

CT is released from V. cholerae and efficiently transported extracellularly by a bacterial type 2 secretion system. The V. cholera secretion system acts like a piston to extrude CT as well as hemagglutinin-protease (HAP) and chitinase into the extracellular space via a pore in the outer membrane. Upon release into the intestinal lumen, CT binds with high affinity and specificity to specific cell membrane receptors. While the ganglioside G _M1 [Gal(β1-3)GalNac(β1-4)(NeuAc(α2-3)Gal(β1-4)Glc] → ceramide was identified many years ago as the receptor for CT, the absolute requirement for binding to this protein was only recently recognized. G _M1 is a glycolipid topped by a branched complex of five sugars including a single sialic acid residue. Each member of the pentameric CTB subunit ring binds to the specific carbohydrate moiety of G _M1 , leading to the interaction of the five subunits with five separate ganglioside G _M1 molecules. Ganglioside G _M1 clusters within detergent insoluble membrane microdomains (lipid raft fractions) of T84 cells and this localization appears to be necessary for toxin internalization as β-methyl-cyclodextrin, a cholesterol chelating agent, prevents CT-mediated Cl ⁻ secretion by blocking CT endocytosis. After binding to GM1, CT is endocytosed by the cell. The specific interaction of CT with G _M1 is critical to toxin internalization. Enterotoxigenic E. coli produce a heat-labile enterotoxin known as LTIIb in which the A subunit functions identically to that of CT while the B subunit preferentially binds to ganglioside G _D1a . Chimeras containing the A subunit of CT and the binding subunits of LTIIb do not cause a secretory response in T84 intestinal epithelial cells despite the availability of G _D1a on the surface. Reciprocally, LTIIb, which is normally nonfunctional in T84 cells, could be rendered active as a chimera containing the A subunit of LTIIb and the B subunits of CT. In human intestinal epithelial cells, lipid raft-mediated endocytosis appears to be the preferred method of entry; however, in other cell types where caveolins are more abundant, caveolae can be utilized for toxin entry. However, it should be noted that these studies were carried out in adult intestinal lines and there is some evidence that there is a substantial difference between the uptake of CT by adult cells versus those of infants. The uptake of CT by fetal small intestinal cells (H4) is significantly greater than by T84 cells and endocytosis occurs through a clathrin-dependent process. Differentiation of these fetal cells using hydrocortisone reverses this phenotype by increasing the association of G _M1 with lipid rafts and inducing internalization via caveolae.

64.2.1.2.1.2

CT Secretion in the Host Cytosol

After endocytosis, CT travels to the endoplasmic reticulum (ER) via a retrograde transport pathway ( Fig. 64.1 ). During the normal process of protein translation and movement outward to the Golgi, some proteins are specifically targeted for retention within the ER by a specific sequence (KDEL). If ER-targeted proteins escape the ER and end up in the Golgi, they are returned to their proper ER location through interaction of the KDEL sequence with an integral membrane protein called ERD2 which shuttles between the ER and Golgi. Because CTA possesses a KDEL sequence in the C-terminus, it resembles a lost ER protein and is promptly escorted to the ER by ERD2. While the KDEL motif enhances the process of retrograde transport, it is not entirely necessary for the process because KDEL mutants of CT are still able to elicit some Cl ⁻ secretion. After arriving in the ER, CT is subject to the actions of protein disulfide isomerase (PDI), which reduces the disulfide bond found between the CT A1 and A2 subunits and assists in the unfolding of the toxin ( Fig. 64.1 ). Much in the way that a space shuttle discards spent booster rockets, the CT A1 subunit can now leave behind the B and A2 subunits as they have served their purpose of binding and transport into the ER. The lone A1 peptide then coopts yet another host system, this time one designed to assist improperly folded proteins traffic from the ER to the cytosol so that they can reach the proteasome and be degraded. This system is referred to as ERAD- or ER-associated protein degradation. The ERAD system uses Sec61, which is normally thought of as an entry channel for newly synthesized proteins. Rather than allowing entry into the ER, the Sec61 complex in conjunction with ERAD allows for the secretion of the CT A1 peptide into the host cytosol. Recent studies utilizing mutant toxin and cell surface biotinylation approaches have shown that CT can in fact breach the intestinal barrier and cross the epithelial cells from the apical to the basolateral surface. These studies indicate an additional mechanism that bypasses retrograde transport to the TGN and enables transport of whole CT probably via a GM1-mediated transcytotic pathway.

Fig. 64.1, Trafficking of cholera toxin (CT) in a polarized epithelial cell. Following attachment of the B subunits of CT to the G M1 ganglioside receptors, the holotoxin is trafficked in a retrograde manner via the Golgi to the endoplasmic reticulum (ER). Through the action of the enzyme, protein disulfide isomerase (PDI), the A1 peptide is released from the A2 peptide, and subsequently enters the cytosol in a Sec61-dependent manner. The A1 peptide ADP ribosylates the G s subunit of adenylate cyclase and locks it in the active state, resulting in an overproduction of cAMP. The ensuing protein kinase A (PKA)-dependent activation of cystic fibrosis transmembrane conductance regulator (CFTR) leads to copious chloride secretion.

64.2.1.2.2 Mechanisms of CT-Associated Diarrhea

64.2.1.2.2.1

cAMP and Secretory Responses

Soon after CT was purified, insights into its mechanism of action were revealed ( Fig. 64.1 ). CT entry into host cytosol and translocation to basolateral membrane allows for the characteristic interaction with Gαs, the regulatory GTPase of adenylate cyclase leading to inhibition of GTP hydrolysis and excessive production of cyclic AMP (cAMP). This step requires CTA1-mediated transfer of the ADP-ribose moiety of NAD ⁺ to an arginine residue (Arg ²⁰¹ ) of an α subunit of Gαs. It is the overproduction of cAMP mediated by CT that triggers Cl ⁻ secretion and fluid imbalance associated with rice-water like diarrhea of V. cholerae . At the time CT was purified and identified, the nature of individual ion transporters and channels was not clearly understood. However, it was noted that addition of either cAMP or CT to rabbit ileal mucosa produced the same effect, an increase in Cl ⁻ secretion and a decrease in Na ⁺ absorption. Therefore, it was clear that alterations in cAMP levels were in fact responsible for the altered secretion resulting from V. cholerae infection. As additional information regarding individual ion channels and transporters was elucidated, a more detailed understanding of the mechanism of cAMP-induced Cl ⁻ secretion emerged. It is now known that the Cl ⁻ channel targeted by CT is CFTR (cystic fibrosis transmembrane conductance regulator). Upon release of cAMP, protein kinase A (PKA) is activated and phosphorylates several key serines in the regulatory domain of CFTR. Phosphorylation of the regulatory domain, along with ATP binding by the two nucleotide-binding domains, results in activation of the channel. The availability of Cl ⁻ for secretion by CFTR is driven by Na ⁺ /K ⁺ ATPase. The opposing action of the Na ⁺ /K ⁺ /2Cl ⁻ cotransporter (NKCC1), which transports K ⁺ , Na ^+, and 2 Cl ⁻ into the cell, and the Na ⁺ /K ⁺ ATPase, which actively secretes Na ⁺ and K ⁺ ions, results in an overabundance of Cl ⁻ within the cell. This excess Cl ⁻ is rectified through the secretion of Cl ⁻ ions. Besides the direct effect of CT on adenylate cyclase activity and cAMP production in enterocytes, CT also stimulates enterochromaffin cells to release the secretagogues serotonin and VIP.

64.2.1.2.2.2

Absorption

While the nature of the comparably smaller decrease in Na ⁺ absorption upon stimulation with CT has been largely ignored in favor of studying Cl ⁻ secretion, it is likely that downregulation of absorptive pathways such as epithelial sodium channel (ENaC) by CFTR is responsible for the decrease in Na ⁺ uptake. In this regard, activation of CFTR via cAMP results in a decrease in ENaC activity. Additionally, CT has been shown to inhibit both NHE2 and NHE3 activity in a cAMP- dependent manner with a more dramatic impact on NHE3. CT has also some effect on a number of aquaporins although they have not been shown to contribute to fluid accumulation in closed loops.

64.2.1.2.3 Human Enteroids as a Model System to Study CT-Associated Diarrhea

The development of a protocol for propagating primary cultures of mouse or human intestine to produce self- organizing mini-intestines, termed enteroids, has provided unique functional models for studying host-pathogen interactions and translating the findings to clinical scenarios. It is interesting to note that O blood group has been associated with more severe cholera infections. A recent study utilizing enteroids demonstrated a direct molecular link between blood group O expression and differential cellular responses to CT. These studies showed that in human enteroids expressing O blood group, CT stimulated robust cAMP relative to enteroids derived from blood group A individuals. In another study, exposure of proximal small intestinal human enteroids to CT-inhibited NHE3 and induced luminal dilatation that is blocked by CFTR inhibitors. Organoid-based functional swelling assays can provide preclinical models to aid in customized drug development and the evaluation of inhibitor potencies of drugs targeting pathogen-derived toxins.

64.2.1.3 Other Toxins of V. cholerae

While CT is well studied and key for altering host physiology during infection, a number of other less understood toxins appear capable of mediating a secretory response or perturbing barrier function that contribute to diarrhea. Because toxins are procured by acquisition of foreign DNA through phage, pathogenicity islands, plasmids, and other mobile genetic elements, not all V. cholerae strains express the same set of toxins. The toxins that affect ion secretion directly include accessory cholera toxin (ACE), which stimulates Ca ^{2 +} -dependent Cl ⁻ secretion; V. cholerae cytolysin (VCC), which creates anion permeable pores and finally, NAG-stable toxin, which activates guanylyl cyclase, thus stimulating cGMP production and leading to PKG-mediated activation of CFTR. The V. cholerae toxins that alter intestinal barrier function include RTX and hemagglutinin/protease or HA/P.

64.2.1.3.1 Ace

Accessory cholera toxin (Ace) was initially discovered as a result of attempts to make a live, attenuated vaccine to prevent cholera. The vaccine strain JBK70 was created by deleting the genes encoding both the A and B subunits of CT. While this strain did not cause the severe diarrhea typically associated with cholera, many of the participants in the study developed mild diarrhea. This suggested that while CT may be the most potent factor contributing to diarrhea, other toxins exist within the parent El Tor strain. By exploring the cholera genome proximal to the CT gene, ctx , referred to as the core region, an additional toxin, Ace was identified. In a strain where the core region containing Ace and CT was deleted, Ace activity was studied by complementing with an Ace-expressing plasmid. Strains expressing Ace increased short circuit current ( I _sc ), a measure of net ion movement, and fluid accumulation in a rabbit ileal loop model. In subsequent in vitro studies using cultured human colonic epithelial T ₈₄ cells, purified Ace elicited a similar increase in I _sc in a Ca ^{2 +} —but not cAMP- or cGMP-dependent manner. The Ace-mediated increase in I _sc was prevented by bumetanide or ouabain, inhibitors of the basally located Na ⁺ /K ⁺ /2Cl ⁻ transporter (NKCC1) and Na ⁺ /K ⁺ ATPase, respectively. Chelation of Ca ^{2 +} with BAPTA-AM or addition of dantrolene, a Ca ^{2 +} antagonist also inhibited the I _sc response as did removal of Cl ⁻ or ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ from the Ringer’s solution in response to Ace. Involvement of a disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS)-sensitive apical Cl ⁻ channel was suspected because the alterations in I _sc were dependent on Cl ⁻ and ${HCO}_{3}^{-}$ ${HCO}_{3}^{-}$ . DIDS caused a 50% reduction in I _sc suggesting that the secretion is mediated by the subsequently identified DIDS-sensitive Ca ^{2 +} -activated Cl ⁻ channel (CaCC).

64.2.1.3.2 VCC

V. cholerae cytolysin (VCC) is a pore-forming toxin that is structurally related to α-toxin and leukocidin F from Staphylococcus aureus . While the S. aureus toxins are known to be heptamers, there is contradictory evidence regarding VCC. Initial biochemical experiments using silver stain suggested that the VCC complex was a pentamer, but subsequent electron microscopic studies suggest that the toxin forms a heptamer as seen for α-toxin and leukocidin-F. The VCC heptamer is believed to be an anion selective channel, in part due to its net positive charge. Bacteria initially secrete VCC in a procytolysin form that must be activated by a cellular protease. Recent studies suggest that the pro-VCC initially binds to the host cell membrane where it encounters ADAM-17, a cellular metalloproteinase that cleaves and thereby activates VCC, which then forms an oligomeric pore. In addition to alterations in ion permeability, VCC causes a dramatic vacuolation phenotype. The anion channel activity of VCC is required for vacuolation as DIDS and SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (disodium salt)) inhibition of the channel prevents this phenotype and cell death. The enlarged vacuoles have more recently been shown to occur as a result of autophagy. During the process of vacuole formation, VCC is internalized and associated with vacuolar membranes. While CT is clearly the most important factor in producing cholera-related diarrhea, VCC increases fluid accumulation in the rabbit ileal loop model and in suckling mice. In addition, VCC elicits a 20% increase in I _sc from human colonic tissue.

64.2.1.4 Heat-Stable Enterotoxin

Heat-stable enterotoxin (NAG-ST or ST) is a 17 amino acid peptide originally characterized in a nonagglutinable (NAG) strain of V. cholerae , so-named because they are not reactive to anti-01 serovar antibodies. NAG-ST acts in a manner similar to E. coli toxin ST _a . NAG-ST induces Ca ^{2 +} release from intracellular stores in response to IP ₃ , which activates PKC-α, leading to the activation of guanylyl cyclase and the production of cGMP. Most of the work performed with heat-stable toxin has been done with E. coli ST _a or EAST-1 and, because of its homology, NAG-ST is presumed to act in a similar manner. The E. coli toxins ST _a and EAST-1 are known to interact directly with membrane-associated guanylyl cyclase C (GC-C) and phosphorylation of guanylyl cyclase by PKC-α enhances its activation and production of cGMP. GC-C receptors mutated to alanine at Ser ¹⁰²⁹ fail to respond to ST _a upon the addition of PMA. Both the activation of PKC-α by NAG-ST and its homology to ST _a suggest that the two toxins act by the same mechanism although a direct interaction of NAG-ST with guanylyl cyclase has not been demonstrated. Production of cGMP activates CFTR through either PKG-II or PKA-II, depending on cell type. Fluid accumulation and I _sc in response to the guanylyl cyclase agonist ST _a were reduced in the small intestine of PKG-II-knockout mice in comparison to control animals. Colon-derived cells such as T84 and Caco-2 also respond to ST _a through activation of PKA-II.

64.2.1.4.1 RTX

RTX toxin, also referred to as VcRtxA, RtxA, or MARTX _vc , elicits cell rounding and loss of epithelial barrier function. This is mediated in part through cross-linking of actin monomers, with a characteristic laddering of actin seen by Western blot. RTX is unusually large at 484 kDa, although only the 48 kDa actin cross-linking domain (ACD; aa 1913–2441) is necessary to mediate actin polymerization. Crystal structure and mass spectrometry of the cross-linked actin revealed residues K50 and E270 to be covalently bound through an isopeptide bond. Toxins lacking the ACD promote an intermediate phenotype of cell rounding without actin polymerization. This effect has been recently attributed to a Rho-inactivating domain (RID) between amino acids 2552–3099. The effect of RID on Rho, Rac, and Cdc42 is not permanent ruling out ADP-ribosylation or degradation and does not mimic GAP or phosphatase activity suggesting a novel mechanism of inactivation. The transepithelial electrical resistance (TER) of T84 monolayers drops significantly without a corresponding increase in I _sc in response to RTX expressing V. cholerae while a RTX mutant strain does not alter TER. These studies were performed in a strain in which HA/P, Ace, and CT had been deleted. In addition, there was a 6.6-fold increase in the permeability of 3000 Da fluorescein-conjugated dextran following infection with the RTX-expressing strain. In addition to the ACD and RID regions of the protein, there is a cysteine protease domain (CPD) that is necessary for autoproteolytic activation of RTX. Interestingly, screens of a cysteine protease inhibitor library identified several candidate inhibitors in the aza-epoxide family that prevent actin cross-linking in human foreskin fibroblasts.

64.2.1.4.2 HA/P

Hemagglutinin/protease or HA/P cleaves a number of substrates including fibronectin, mucin, and lactoferrin in addition to the nick site necessary for activation of CT. There was a 90% drop in TER in T84 monolayers infected with a V. cholerae strain deleted for the core region of the CT-containing phage, which harbors CT, Ace, and RtxA (Bah-3). In contrast, an isogenic deletion mutant in hapA had no effect on TER. Complementation of this mutant fully restored the ability to disrupt barrier function. This work represents molecular confirmation of the earlier work by Wu et al., which showed that HA/P decreased TER in MDCK monolayers. A later study by the same group suggested that the loss of barrier function was due to both degradation of occludin and relocation of ZO-1. HA/P is an extracellular toxin and degrades occludin only at the two extracellular domains creating two antibody-reactive products of 35 and 50 kDa, which differ from those seen with trypsin degradation. The degradation of occludin was prevented by the zinc metalloprotease inhibitor Zincov as well. Changes in occludin were preceded by the disappearance of ZO-1 from tight junctions; however, ZO-1 was not degraded.

64.2.2 Vibrio parahemolyticus

Another member of the Vibrio family, V. parahemolyticus , is predominantly found in brackish coastal waters and causes a limited number of human infections per year. These infections are typically acquired through the consumption of raw oysters and during outbreaks harvest is postponed in heavily contaminated oyster beds until cooler waters, less favorable to V. parahemolyticus growth, arrive. Pathogenic V. parahemolyticus is characterized by production of thermostable direct hemolysin (TDH); however, it should be noted that < 1% of environmental isolates actually contain the TDH toxin. In addition to TDH, V. parahemolyticus produces another toxin known as TDH-related hemolysin (TRH). TRH is similar to TDH, but is incapable of creating the characteristic β-hemolysis on Wagatsuma agar, typically used for identifying pathogenic isolates of this species. TDH and TRH appear to be structurally and functionally similar and therefore the better-studied TDH will be used as an example.

There are several variants of the tdh gene that are associated with insertion elements within the V. parahemolyticus chromosome although tdh2 appears to be primarily responsible for the classic hemolytic phenotype. The structure of TDH is not well defined although the mature toxin functions as a dimer and contains an intramolecular disulfide bond between Cys ¹⁵¹ and Cys ¹⁶¹ . TDH activates PKC and raises intracellular calcium levels leading to a Cl ⁻ -mediated increase in I _sc . The DIDS and Ca ^{2 +} sensitivity of the subsequent Cl ⁻ secretion suggests involvement of the calcium-activated chloride channel (CaCC). Correspondingly, inhibition of CFTR with glibenclamide failed to alter the TDH-induced I _sc response.

In addition to TDH and TRH, two type III secretion systems (T3SS) have been recognized in V. parahemolyticus . They reside on two different chromosomes and are labeled I and II accordingly. T3SS I affects cell rounding and tight junctions and will be discussed in the second half of this chapter while T3SS II has both cytotoxic and enterotoxic effects. Studies of this T3SS were only recently initiated; however; mutations that prevent delivery of translocated effector proteins such as VopT, also prevent cAMP stimulation and fluid accumulation in rabbit ileal loops. T3SS II had more of an impact than TDH in infected rabbit ileal loops. Loop inflation decreased 75% in a T3SS II mutant but was not changed by mutation of T3SS I. Thus, type 3 secreted effectors are important elements and perhaps even primary factors in V. parahemolyticus infections.

V. parahemolyticus also has an effect on tight junctions. TER decreased 80% by 3 h postinfection and caused extensive mislocalization of ZO-1 in a manner independent of TDH. While a specific mechanism for these tight junction changes has not been identified, one effector, VopS, has been shown to AMPylate the Rho family of GTPases, preventing their activity. The rapid loss in TER and ZO-1 phenotype are consistent with the inactivation of small G-proteins by TcdA and B. In addition, VopS expression in HeLa cells promoted cell rounding. VopS (Vp1686) is secreted by T3SS I and causes Rho family inhibition.

64.2.3 Enterotoxigenic E. coli

ETEC causes traveler’s secretory diarrhea characterized by massive intestinal fluid secretion similar to cholera that may range from mild to life threatening. The key virulence attributes of ETEC include adherence to epithelial cell surfaces by colonization factors and elaboration of heat-labile (LT) and heat-stable (ST _a ) enterotoxins. Some strains of ETEC may also express enteroaggregative heat-stable toxin 1 (EAST1).

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