Asaomi Kuwae

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1.

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule‐bound GEF‐H1, a RhoA‐specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG‐ and Orf3‐induced stress fiber formation was inhibited by the expression of dominant‐negative forms of GEF‐H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA–ROCK signaling pathway via GEF‐H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF‐H1.

Enteropathogenic Escherichia coli Type III Effectors EspG and EspG2 Alter Epithelial Paracellular Permeability

ABSTRACT Enteropathogenic Escherichia coli (EPEC) delivers a subset of effectors into host cells via a type III secretion system. Here we show that the type III effector EspG and its homologue EspG2 alter epithelial paracellular permeability. When MDCK cells were infected with wild-type (WT) EPEC, RhoA was activated, and this event was dependent on the delivery of either EspG or EspG2 into host cells. In contrast, a loss of transepithelial electrical resistance and ZO-1 disruption were induced by infection with an espG/espG2 double-knockout mutant, as was the case with the WT EPEC, indicating that EspG/EspG2 is not involved in the disruption of tight junctions during EPEC infection. Although EspG- and EspG2-expressing MDCK cells exhibited normal overall morphology and maintained fully assembled tight junctions, the paracellular permeability to 4-kDa dextran, but not the paracellular permeability to 500-kDa dextran, was greatly increased. This report reveals for the first time that a pathogen can regulate the size-selective paracellular permeability of epithelial cells in order to elicit a disease process.

Assembly of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

Bordetella evades the host immune system by inducing IL-10 through a type III effector, BopN.

The inflammatory response is one of several host alert mechanisms that recruit neutrophils from the circulation to the area of infection. We demonstrate that Bordetella, a bacterial pathogen, exploits an antiinflammatory cytokine, interleukin-10 (IL-10), to evade the host immune system. We identified a Bordetella effector, BopN, that is translocated into the host cell via the type III secretion system, where it induces enhanced production of IL-10. Interestingly, the BopN effector translocates itself into the nucleus and is involved in the down-regulation of mitogen-activated protein kinases. Using pharmacological blockade, we demonstrated that BopN-induced IL-10 production is mediated, at least in part, by its ability to block the extracellular signal-regulated kinase pathway. We also showed that BopN blocks nuclear translocation of nuclear factor κB p65 (NF-κBp65) but, in contrast, promotes nuclear translocation of NF-κBp50. A BopN-deficient strain was unable to induce IL-10 production in mice, resulting in the elimination of bacteria via neutrophil infiltration into the pulmonary alveoli. Furthermore, IL-10–deficient mice effectively eliminated wild-type as well as BopN mutant bacteria. Thus, Bordetella exploits BopN as a stealth strategy to shut off the host inflammatory reaction. These results explain the ability of Bordetella species to avoid induction of the inflammatory response.

BopC Is a Novel Type III Effector Secreted by Bordetella bronchiseptica and Has a Critical Role in Type III-dependent Necrotic Cell Death

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.

BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild‐type strain. The results showed that the wild‐type strain, but not the type III mutant, secreted a 40‐kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N‐terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild‐type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild‐type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild‐type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.

Differential Expression of Type III Effector BteA Protein Due to IS481 Insertion in Bordetella pertussis

Background Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s) involved in the type shift. Methodology/Principal Finding Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 5′ untranslated region of bteA, −136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion. Conclusion/Significance The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis.

The Type III Secreted Protein BopD in Bordetella bronchiseptica Is Complexed with BopB for Pore Formation on the Host Plasma Membrane

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B. bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.

The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica.

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron‐depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron‐replete conditions. Furthermore, it was confirmed that induction of T3SS‐dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron‐depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron‐depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.