Fluorescence can be used to trace the fate of exogenous micro-organisms inside the alimentary tract of mosquitoes

There is a great deal of current research interest in utilising bacteria for the control of intractable arthropod-borne diseases such as dengue. Although there is accumulating evidence that bacterial infection is a promising control strategy, most studies on bacteria-insect interactions lacked useful markers for detecting pathogenesis. This provided the impetus to investigate bacterial infection in the dengue vector Aedes albopictus. The infection persistence patterns in key organs of the alimentary canal of females were examined using a GFP-expressing strain of Escherichia coli (Migula). Just after feeding with sugar meal containing the bacteria, the crop and midgut as well as parts of the Malpighian tubules showed fluorescence. From 1 h onwards, bacterial populations declined sharply in both the midgut and crop, with complete elimination in the former but persistence of bacteria at 7 h post-feeding in the latter. After 24 h, neither organ retained the fluorescent marker. However, culture of homogenates of these organs in Luria-Bertani medium revealed the presence of a bacterial population in the crop, but not in the midgut. These observations suggest a difference in the potential hysiological actions expressible by the two organs. In fact, both are storage sites for ingested fluids, but the midgut has greater physiological activity. Presumably, one of these activities contributed to eliminating GFPexpressing E. coli from the A. albopictus midgut after 24 h. The results of the present study using a fluorescent marker to detect infection may be useful for developing strategies to fully characterise the main steps involved in the bacterial infection process in insects

Elevation of dopamine level reduces hostseeking activity in the adult female mosquito Aedes albopictus

Background: Mosquito-borne viruses are transmitted to human hosts via blood-feeding behavior of female mosquitoes. Female mosquitoes seek a host to take blood meals (host-seeking behavior). In order to prevent virus infections, it is important to understand how they modulate host-seeking behavior. Dopamine (DA) in the central nervous system acts as a neuromediator that regulates a variety of behaviors in insects. In female mosquitoes, host-seeking behavior increases when DA levels in the head decline after emergence. However, it remains unclear whether DA directly modulates host-seeking behavior in female mosquitoes. The aim of this study was to examine whether changes in DA levels in the head affects host-seeking activity in the adult female mosquito Aedes albopictus (Ae. albopictus). Findings: We compared host-seeking behavior in one group of emerging female adults treated with L-β-3,4-dihydroxyphenylalanine (L-DOPA), the precursor of DA, (L-DOPA group), with that in an untreated control (control group) after confirming elevation of head DA in L-DOPA group by using high-performance liquid chromatography. The content of head DA in L-DOPA group significantly remained higher than that in controls on all days examined. The host-seeking activity in the control group showed a gradual increase over the 6-day experimental period. In contrast, there was no such increase in the host-seeking activity in the L-DOPA group. Therefore, the host-seeking activity of L-DOPA group was significantly lower than that of the controls between day 3 and 6 post-emergence. Conclusion: Our results indicate that elevation of DA level reduces host-seeking activity in adult female mosquito Ae. albopictus

Tyrosine phosphorylation of p62(dok) by p210(bcr-abl) inhibits RasGAP activity

The t(9;22) chromosomal translocation is found in almost all patients with chronic myelogenous leukemia. The resultant Bcr-Abl fusion gene expresses a chimeric fusion protein p210(bcr-abl) with increased tyrosine kinase activity. Hematopoietic progenitors isolated from chronic myelogenous leukemia patients in the chronic phase contain constitutively tyrosine-phosphorylated p62(dok) protein. p62(dok) associates with the Ras GTPase-activating protein (RasGAP), but only when p62(dok) is tyrosine phosphorylated. Here we have investigated the interaction between p62(dok) and RasGAP and the consequences of p62(dok) tyrosine phosphorylation on the activity of RasGAP. We have found that p62(dok) is directly tyrosine phosphorylated by p210(bcr-abl), and the sites of phosphorylation are located in the C-terminal half of the p62(dok) molecule. We have identified five tyrosine residues that are involved in in vitro RasGAP binding and have found that tyrosine-phosphorylated p62(dok) inhibits RasGAP activity. Our results suggest that p210(bcr-abl) might lead to the activation of the Ras signaling pathway by inhibiting a key down-regulator of Ras signaling

Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

BACKGROUND: Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. RESULTS: We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability. CONCLUSIONS: The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs