Inflammation and Disintegration of Intestinal Villi in an Experimental Model for Vibrio parahaemolyticus-Induced Diarrhea

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis in many parts of the world, but there is limited knowledge of the pathogenesis of V. parahaemolyticus-induced diarrhea. The absence of an oral infection-based small animal model to study V. parahaemolyticus intestinal colonization and disease has constrained analyses of the course of infection and the factors that mediate it. Here, we demonstrate that infant rabbits oro-gastrically inoculated with V. parahaemolyticus develop severe diarrhea and enteritis, the main clinical and pathologic manifestations of disease in infected individuals. The pathogen principally colonizes the distal small intestine, and this colonization is dependent upon type III secretion system 2. The distal small intestine is also the major site of V. parahaemolyticus-induced tissue damage, reduced epithelial barrier function, and inflammation, suggesting that disease in this region of the gastrointestinal tract accounts for most of the diarrhea that accompanies V. parahaemolyticus infection. Infection appears to proceed through a characteristic sequence of steps that includes remarkable elongation of microvilli and the formation of V. parahaemolyticus-filled cavities within the epithelial surface, and culminates in villus disruption. Both depletion of epithelial cell cytoplasm and epithelial cell extrusion contribute to formation of the cavities in the epithelial surface. V. parahaemolyticus also induces proliferation of epithelial cells and recruitment of inflammatory cells, both of which occur before wide-spread damage to the epithelium is evident. Collectively, our findings suggest that V. parahaemolyticus damages the host intestine and elicits disease via previously undescribed processes and mechanisms.     

Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and β-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.     

The role of type III secretion System 2 in Vibrio parahaemolyticus pathogenicity

Vibrio parahaemolyticus, a Gram-negative marine bacterial pathogen, is emerging as a major cause of food-borne illnesses worldwide due to the consumption of raw seafood leading to diseases including gastroenteritis, wound infection, and septicemia. The bacteria utilize toxins and type III secretion system (T3SS) to trigger virulence. T3SS is a multi-subunit needle-like apparatus used to deliver bacterial proteins, termed effectors, into the host cytoplasm which then target various eukaryotic signaling pathways. V. parahaemolyticus carries two T3SSs in each of its two chromosomes, named T3SS1 and T3SS2, both of which play crucial yet distinct roles during infection: T3SS1 causes cytotoxicity whereas T3SS2 is mainly associated with enterotoxicity. Each T3SS secretes a unique set of effectors that contribute to virulence by acting on different host targets and serving different functions. Emerging studies on T3SS2 of V. parahaemolyticus, reveal its regulation, translocation, discovery, characterization of its effectors, and development of animal models to understand the enterotoxicity. This review on recent findings for T3SS2 of V. parahaemolyticus highlights a novel mechanism of invasion that appears to be conserved by other marine bacteria.     

Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway

Vibrio parahaemolyticus is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of V. parahaemolyticus. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway.     

The <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis

Background  

Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.     

The BtaF Trimeric Autotransporter of Brucella suis Is Involved in Attachment to Various Surfaces,Resistance to Serum and Virulence

The adhesion of bacterial pathogens to host cells is an event that determines infection, and ultimately invasion and intracellular multiplication. Several evidences have recently shown that this rule is also truth for the intracellular pathogen Brucella. Brucella suis displays the unipolar BmaC and BtaE adhesins, which belong to the monomeric and trimeric autotransporter (TA) families, respectively. It was previously shown that these adhesins are involved in bacterial adhesion to host cells and components of the extracellular matrix (ECM). In this work we describe the role of a new member of the TA family of B. suis (named BtaF) in the adhesive properties of the bacterial surface. BtaF conferred the bacteria that carried it a promiscuous adhesiveness to various ECM components and the ability to attach to an abiotic surface. Furthermore, BtaF was found to participate in bacterial adhesion to epithelial cells and was required for full virulence in mice. Similar to BmaC and BtaE, the BtaF adhesin was expressed in a small subpopulation of bacteria, and in all cases, it was detected at the new pole generated after cell division. Interestingly, BtaF was also implicated in the resistance of B. suis to porcine serum. Our findings emphasize the impact of TAs in the Brucella lifecycle.     

Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus‐associated adhesin

Tissue damage predisposes humans to life‐threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibres. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus‐associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fibre retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.     

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height,Cell-Substrate Interactions,and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell''s height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.     

Autophagy Controls an Intrinsic Host Defense to Bacteria by Promoting Epithelial Cell Survival: A Murine Model

Cell death is a critical host response to regulate the fate of bacterial infections, innate immune responses, and ultimately, disease outcome. Shigella spp. invade and colonize gut epithelium in human and nonhuman primates but adult mice are naturally resistant to intra-gastric Shigella infection. In this study, however, we found Shigella could invade the terminal ileum of the mouse small intestine by 1 hour after infection and be rapidly cleared within 24 h. These early phase events occurred shortly after oral infection resulting in epithelial shedding, degranulation of Paneth cells, and cell death in the intestine. During this process, autophagy proceeded without any signs of inflammation. In contrast, blocking autophagy in epithelial cells enhanced host cell death, leading to tissue destruction and to inflammation, suggesting that autophagic flow relieves cellular stress associated with host cell death and inflammation. Herein we propose a new concept of “epithelial barrier turnover” as a general intrinsic host defense mechanism that increases survival of host cells and inhibits inflammation against enteric bacterial infections, which is regulated by autophagy.     

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.     

PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10² ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10³ cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.     

In Vitro Protective Efficacy of Clostridium butyricum Against Fish Pathogen Infections

Most pathogens in intestine are opportunist, called “opportunistic pathogens” that usually do not cause disease in a healthy host. Only when the host’s resistance is lowered or the intestinal microecological balance is destroyed, the opportunistic pathogens are capable of causing disease. Here, two opportunistic pathogens, Salmonella enteritidis and Vibrio parahaemolyticus were chosen to test the possible antagonistic effect of the probiotic agent Clostridium butyricum on these pathogens infections in vitro using fish intestinal epithelial cells (FIECs). The C. butyricum and its spent culture supernatants exhibited significant inhibitory activity on S. enteritidis and V. parahaemolyticus growth and adherence to FIECs. The C. butyricum also showed significant inhibitory effects on S. enteritidis and V. parahaemolyticus induced apoptosis, which may due to its growth and adhesion inhibitory effects. These results indicated that the probiotic bacterium C. butyricum has preventive and therapeutic effects on S. enteritidis and V. parahaemolyticus infections in fish.     

The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella sonnei pathogenesis

Shigella sonnei is a bacterial pathogen and causative agent of bacillary dysentery. It deploys a type III secretion system to inject effector proteins into host epithelial cells and macrophages, an essential step for tissue invasion and immune evasion. Although the arsenal of bacterial effectors and their cellular targets have been studied extensively, little is known about the prerequisites for deployment of type III secreted proteins during infection. Here, we describe a novel S. sonnei adhesin, SSO1327 which is a multivalent adhesion molecule (MAM) required for invasion of epithelial cells and macrophages and for infection in vivo. The S. sonnei MAM mediates intimate attachment to host cells, which is required for efficient translocation of type III effectors into host cells. SSO1327 is non‐redundant to IcsA; its activity is independent of type III secretion. In contrast to the up‐regulation of IcsA‐dependent and independent attachment and invasion by deoxycholate in Shigella flexneri, deoxycholate negatively regulates IcsA and MAM in S. sonnei resulting in reduction in attachment and invasion and virulence attenuation in vivo. A strain deficient for SSO1327 is avirulent in vivo, but still elicits a host immune response.     

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet‐activating factor receptor

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host–pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet‐activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community‐acquired pneumonia in the elderly, we explored the role of A1 in the age‐driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial–host interactions.     

Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is the most common cause of bacterial, seafood‐related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro‐ and anti‐inflammatory cytokines by V. parahaemolyticus‐infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL‐1α, IL‐6, TNF‐α and IL‐10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T‐cell co‐stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.     

Salmonella Typhimurium Type III Secretion Effectors Stimulate Innate Immune Responses in Cultured Epithelial Cells

Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP) kinase and NF-κB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.