An evaluation of fusion partner proteins for paratransgenesis in Asaia bogorensis.

Mosquitoes transmit many pathogens responsible for human diseases, such as malaria which is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are increasingly undermined by mosquito and pathogen resistance, so additional methods of control are required. Paratransgenesis is a method whereby symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. Previously, engineered Asaia strains using native signals to drive the release of the antimicrobial peptide, scorpine, fused to alkaline phosphatase were successful in significantly suppressing the number of oocysts formed after a blood meal containing P. berghei. However, these strains saw high fitness costs associated with the production of the recombinant protein. Here, we report evaluation of five different partner proteins fused to scorpine that were evaluated for effects on the growth and fitness of the transgenic bacteria. Three of the new partner proteins resulted in significant levels of protein released from the Asaia bacterium while also significantly reducing the prevalence of mosquitoes infected with P. berghei. Two partners performed as well as the previously tested Asaia strain that used alkaline phosphatase in the fitness analyses, but neither exceeded it. It may be that there is a maximum level of fitness and parasite inhibition that can be achieved with scorpine being driven constitutively, and that use of a Plasmodium specific effector molecule in place of scorpine would help to mitigate the stress on the symbionts.

Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi.

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are hindered by mosquito and pathogen resistance, so research has turned to altering the microbiota of the vectors. In this strategy, called paratransgenesis, symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver antiplasmodial effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. However, common secretion signals from E. coli and closely related species do not function in Asaia. Here, we report evaluation of 20 native Asaia N-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of antiplasmodial effector molecules directed to the periplasm and ultimately outside the cell. We tested the hypothesis that by increasing the amount of antiplasmodials released from the cell we would also increase parasite killing power. We scanned the Asaia bogorensis SF2.1 genome to identify signal sequences from extra-cytoplasmic proteins and fused these to the reporter protein alkaline phosphatase. Six signals resulted in significant levels of protein released from the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three Asaia strains were able to suppress the number of oocysts formed after a blood meal containing P. berghei to a significantly greater degree than wild-type Asaia, although prevalence was not decreased beyond levels obtained with a previously isolated siderophore receptor signal sequence. We interpret these results to indicate that there is a maximum level of suppression that can be achieved when the effectors are constitutively driven due to stress on the symbionts. This suggests that simply increasing the amount of antiplasmodial effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains and that symbiont fitness must be considered as well.

Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood-Brain Barrier after Traumatic Brain Injury.

Neuroinflammation and dysfunction of the blood-brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the in vitro model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI.