Thus, low affinity Tulathromycin B interactions might have escaped our detection, but might still have relevance in vivo, if the local concentrations of the interactors are sufficiently high. Notwithstanding the above considerations, a number of controls support the notion that we should have obtained a near complete representation of the EH interactome for EHS-1, ITSN-1 and REPS-1. Conversely, we may have missed a number of interactions for the EH of RME-1, because of the nature of our screening. It has been shown that homo/hetero-oligomerization of EHD proteins is important for optimal binding to NPF-containing proteins, a condition that most likely was not achieved under the conditions of our initial Y2H screening, thus preventing the isolation of strong specific interactors. This is further supported by the fact that the EH domain of RME-1/EHD proteins, located in the carboxylterminal of the proteins, has a strong binding preference for NPF motifs followed by acidic residues. None of the proteins identified in our Y2H screens show an acidic consensus surrounding the NPF motif, suggesting that the RME-1 EH binding proteins we identified are probably promiscuous interactors. Indeed, the described interaction between AMPH-1 and RME-1, which was previously shown to be functionally relevant, was not identified in our screening. Regardless of the conditions of screening, it is of note that 14 of the 26 genes encoding for EH-interactors displayed genetic interactions with at least one gene encoding an EH-containing protein. This is remarkable, considering that only one phenotype was analyzed. While a number of these interactions were already known, either in nematodes or in mammals, the others are described here for the first time : together, these interactions define the physical and functional landscape of the EH network at the organismal level in the nematode. As shown in Figure 5, the most evident feature of the EH network is its involvement in LOUREIRIN-B endocytosis, traffic, and actin dynamics. These results confirm the role of the EH network in orchestrating processes in which coordination between the machineries of intracellular traffic and actin remodeling are required. This function is evolutionarily conserved: it has been confirmed in a number of high-resolution studies in mammals, and also by a virtual reconstruction of the EH network in yeast, which we performed by exploiting a number of publicly available interaction data and published high-throughput screens in S. cerevisiae. At the biological level, the EH network seems to play a major role in neurotransmission in the nematode, as supported by the finding that RNAi of the majority of EH interactors affected aldicarb sensitivity either in a WT background or in EH containing proteins mutant strains. While these results can probably be interpreted in the framework of the known participation of EH-containing proteins to the process of synaptic vesicle recycling, through the mentioned connections with endocytosis/traffic and actin dynamics, there is reason to postulate a wider involvement of the EH network in neurotransmission. In particular, the involvement of the EH network in the physiological regulation of the nervous system might also be mirrored by its putative subversion in pathological conditions. Indeed, some of the mammalian homologues of the EHinteracting proteins we identified in the nematode have been implicated in Alzheimer’s disease.

Three of the four EH-containing nematode proteins and genes, EHS-1, ITSN-1, and RME-1 have been previously characterized at high resolution. However, REPS-1 and its gene, reps-1, remain uncharacterized. Thus, we therefore performed a preliminary characterization of REPS-1. A mutant strain, reps-1, was obtained from the National Bioresearch Project. reps-1 is predicted to encode for a protein of 410 amino acids and its genomic organization is presented in Figure 3A. The tm2156 mutant allele has a deletion of 779 bases resulting in loss of the third intron and of a portion of the fourth exon. reps-1 animals appear to be wild type at different temperatures, in terms of viability, fertility and locomotion. To gain insight into reps-1 functions, we analyzed its expression pattern using transgenic lines carrying the reps-1 gene under its own promoter, in fusion with a GFP reporter. The expression of the fusion protein was analyzed in lysates of transgenic worms by western blot analysis, revealing a protein band with an apparent molecular weight of 75 kDa, in agreement with the predicted molecular weight for REPS-1::GFP. The transgenic lines showed expression in many tissues including intestine, secretory system, vulval cells and muscle cells. REPS-1 was also expressed in the nervous system with diffuse staining in the nerve ring, ventral cord and commissures, but no expression was observed in the neuronal body. When tested for sensitivity to aldicarb, an inhibitor of acetylcholine esterase often used to reveal defective cholinergic transmission, the reps-1 mutant showed an abnormal response, with hypersensitivity to the drug LOUREIRIN-B compared to wild type animals, a phenotype reminiscent of that detected in itsn-1-null nematodes. The aberrant response to aldicarb that may be related to deficiencies at neuronal and/or muscular levels, where REPS-1 is expressed, strongly suggests a role of REPS-1 in neurotransmission. This result does not exclude, obviously, other possible 4-(Benzyloxy)phenol functions for REPS-1, as also suggested by the wide pattern of expression of the gene. The physical and functional connections in the EH network of the nematode are reported in schematic form in Figure 5 and in an extended form in Figure S4; in addition, we report a number of characteristics of the identified EH interactors as obtained from literature searches and Wormbase. We identified 26 interactors of EH domains by Y2H and validated a majority of them through in vitro binding assays and by genetic analysis. We cannot be certain that we have identified all EH-interacting proteins. Few hypothetical interactors, as for example the synaptojanin homologue UNC-26, were unable to interact with the EH baits, even when directly tested. This might be due to “real” lack of interaction or to technical reasons. For instance, the absence �C in the EH constructs used for the screening �C of regions outside of the EH domain required to assist some EH-NPF interactions might have yielded a false negative result. It should also be mentioned that the nature of our screening does not allow for stringent conclusions in terms of affinity of the detected interactions. It is known that several variables affect the affinity and the selectivity of EH-NPF interactions, such as the amino acid composition of NPF surrounding regions.

Thus, by nature, cell migration is a process of global Butenafine hydrochloride reorganization of cytoskeleton. For example, actin polymerization drives the formation and extension of the protrusions such as lamellipodia at the leading edge, while the asymmetric distribution and enzymatic engagement of myosin and actin produce the force for cellular contractility and lead to the retraction of the trailing edge. Rho small GTPases are clearly pivotal in all of these cytoskeletal rearrangement processes. For example, Rac is primarily responsible for generating a protrusive force through the localized actin polymerization, while Rho is responsible for the Tulathromycin B contraction of the cell body and the retraction of the rear end. As downstream effectors of Rho GTPases, cofilin severs actin filament to generate barbed ends and thus facilitates the actin treadmilling while Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The severing activity of cofilin and branching activity of Arp2/3 function coordinately to promote the formation of a branched actin network or cortical actin meshwork at the leading edge and generate propulsive force for migrating cells. The activation of Rho kinase, an effector of RhoA, leads to increased myosin phosphorylation and actomyosin contractility and therefore is required for the retraction process during cell movement. The profound morphological changes induced by KAI1/CD82 apparently resulted from the aberrant organization and/or reorganization of cellular cytoskeleton networks. Actin is the cytoskeleton system that drives the cell movement-related subcellular events including protrusion, traction, and retraction. As predicted, actin cytoskeleton became globally aberrant upon KAI1/CD82 overexpression. The reduced or deficient cortical meshwork and stress fibers in KAI1/CD82 overexpressing cells suggested the aberrancy in actin polymerization. Such aberrancy can be further exacerbated by the reduced integrin signaling in KAI1/CD82-overexpressing cells. The development of cortical actin network generates protrusive force, morphologically revealed as the lamellipodia formation. Therefore, the formation of actin cortical meshwork and simultaneous extension of leading lamellipodia are the major subcellular morphological features of many migrating cells. In KAI1/CD82-overexpressing Du145 cells, the loss of these morphological characteristics was apparently caused by aberrant actin organization, particularly the polymerization of branched, cortical actin meshwork. In parallel, the retraction process is also essential for the movement of many types of cells. The defects in retraction upon KAI1/CD82 expression were displayed as either elongated trailing tails when Du145-KAI1/CD82 cells were treated with HGF or spread on LN1-coated plates, long cellular extensions in the cells with bipolar or dumbbell shape, or persistent vertices in cells with irregular polygonal shape. Mechanistically, the deficiency in retraction could result from an abnormality of actinmyosin retraction machinery. In the case of KAI1/CD82, the decreased ROCK activity caused functional incompetence of this machinery while the fewer stress fibers made the retraction process lose its structural base. Rho small GTPases are the master regulators of actin reorganization. Rac activation stimulates membrane ruffling through polymerization of cortical actin near the cell periphery, while Rho activation stimulates cell contractility through assembly of mainly radial-oriented actin stress fibers.

Thus, it is possible that the immunization regimen induced an immune response that prevented a systemic dissemination of the bacteria. Studies by Sharma et al. provide support for this possibility. It is also possible that high titers of antigen-specific IgG, as well as cellmediated immune responses in the systemic compartment helped control bacterial growth in the spleen and liver. In the case of the lungs, it might be more difficult to clear the infection, especially considering the high number of bacteria that are rapidly lodged in the lungs after i.n. infection. Since the subunit vaccine used in this current study was tested for its efficacy in inducing a protective response against FT LVS and not against the Schu S4 strain, the question of the 4-(Benzyloxy)phenol degree of similarity between FT LVS and Schu S4-derived DnaK and Tul4 needs to be addressed. Analysis of the outer membrane proteins of FT LVS and Schu S4 has revealed almost identical bioinformatics findings and a shared sequence homology of 96 to 100%, which was confirmed with antiserum reactivity to homologous outer membrane proteins, such as Tul4, found in FT LVS and Schu S4. In a different study, DnaK was among the 3,4,5-Trimethoxyphenylacetic acid identified proteins to which T cell hybridomas showed select reactivity upon exposure to FT LVS and Schu S4 lysates. Moreover, the protein sequences encoding the DnaK and Tul4 T cell epitopes are conserved in FT LVS and Schu S4. Hence, the degree of similarity between the DnaK and Tul4 derived from FT LVS and Schu S4 is extremely high. Based upon these observations, we predict immune reactivity to DnaK and Tul4 in mice immunized with both DnaK+Tul4 will contribute towards a host defense against FT Schu S4 infection. However, it should be kept in mind that the levels of protein expression by a bacterium could influence the efficacy of a vaccine. Studies have shown that the expression of proteins by strains of FT can vary based upon growth conditions, e.g., laboratory media, host macrophages or murine spleens. Therefore, if FT Schu S4 exhibits low levels of DnaK and/or Tul4 expression, especially during the in vivo infection process, this could potentially limit any protective effects of a subunit vaccine. Future studies will be required to investigate this possibility and to determine the effectiveness of our subunit vaccine in protecting against a lethal challenge with FT Schu S4. Studies have shown that a protective response against FT LVS challenge does not necessarily translate into an effective response against a Schu S4 challenge. For example, immunization with the outer membrane protein FopA was effective against FT LVS, but not a Schu S4 challenge. In another study native outer membrane proteins afforded 50%, but not complete protection against a Schu S4 challenge. Similarly, LPS has been extensively shown to protect mice against FT LVS infection, but a response to LPS alone is not sufficient to completely protect against a type A strain. These findings add to the growing consensus that in order to provide effective protection against more virulent FT subspecies, immune reactivity to several FT antigens will be necessary. Moreover, a combinatorial strategy, where LPS is chemically conjugated to a protein antigen such as bovine serum albumin, significantly increased protection against a type A strain. This highlights the need to identify a diverse set of FT-specific immunodominant antigens that can stimulate antibody and cell-mediated responses to combat FT infection.

In a different study, FopA, another outer membrane protein, was shown to protect mice against FT LVS infection, and antibodies played an important role. Moreover, it is Tulathromycin B possible that Tul4 may play a role in the adherence of FT to lung cells, and therefore, mucosal IgA anti-Tul4 antibodies may limit bacterial colonization and promote clearance. In this regard, IgA antibodies to FT have been shown to be important for protection against respiratory tularemia. Tul4 is a TLR2 agonist. Therefore, in addition to the adjuvant effects of GPI, it is possible that Tul4 might also act in potentiating innate and/or adaptive immune responses. In this regard, it has been shown that innate immune responses induced via several TLRs can protect against a lethal FT challenge : however, for optimal protection, TLR agonists need to be administered 1�C3 days before infection. Furthermore, since TLRs influence adaptive immune responses; it is possible that Tul4mediated TLR signaling might provide additional priming for the development of an antigen-specific response. This possibility gains support from the evidence that Tul4 induces a robust antibody response even in the absence of an adjuvant, and from previous studies demonstrating the importance of TLR2 in host responses to F. tularensis. Cytokines are important players of immune responses, and several cytokines, including TNF-a, IFN-c, IL-6, IL-12, IL-17A and IL-10, have been shown to be relevant in the host defense against FT infection. However, our studies did not reveal a specific cytokine pattern associated with protection seen in DnaK+Tul4+GPI immunized mice. In this regard, no difference was observed in the level or pattern of the IFN-c response among all experimental groups of mice, even though IFN-c has been shown to be a critical cytokine in host responses against FT infection. Moreover, between 3�C5 days after infection, the levels of IL-12p70, TNF-a and IL-6 were higher in mice immunized with one of the antigens than in mice immunized with both DnaK and Tul4, a pattern similar to that seen with the other cytokines assessed. These findings suggest that in vivo, protection against FT infection likely occurs due to the presence and interactions of numerous inflammatory mediators. Molecules like IP-10, CXCL1, RANTES and G-CSF that were evaluated in the present study are also critical participants in immune responses. For instance, RANTES, IP-10 and CXCL1, are chemotactic cytokines that belong to the chemokine family and play active roles in recruiting leukocytes to inflammatory sites. Furthermore, G-CSF, besides being a growth factor, is a cytokine that stimulates the survival, proliferation, differentiation and function of neutrophils, in addition to its involvement in the regulation of the PI3K/Akt signaling pathway. Finally, the observed changes in cytokine levels between 3 to 5 days after infection are perhaps important for the observed protection, since a differential modulation of the cytokines during this period was observed between infected mice immunized with each antigen and mice immunized with the LOUREIRIN-B vaccine preparation containing both antigens. Five days post i.n. infection of mice with FT LVS, the relative bacterial burden in the livers and spleens was roughly similar to that observed in the lungs of non-immunized mice. This finding indicates that there was a notable systemic dissemination of the bacteria, as has been reported earlier. However, there was a significant decrease in the relative bacterial burden in the lungs, livers and spleens of immunized compared to non-immunized mice.