Vector-borne diseases are a substantial portion of the global disease burden; one of the deadliest of these is malaria. Vector control strategies have been hindered by mosquito and pathogen resistances, and population alteration approaches using transgenic mosquitos still have many hurdles to overcome before they can be implemented in the field. Here we report a paratransgenic control strategy in which the microbiota of Anopheles stephensi was engineered to produce an antiplasmodial effector causing the mosquito to become refractory to Plasmodium berghei. The midgut symbiont Asaia was used to conditionally express the antiplasmodial protein scorpine only when a blood meal was present. These blood meal inducible Asaia strains significantly inhibit pathogen infection, and display improved fitness compared to strains that constitutively express the antiplasmodial effector. This strategy may allow the antiplasmodial bacterial strains to survive and be transmitted through mosquito populations, creating an easily implemented and enduring vector control strategy.

Asaia spp. are abundant members of the microbiota of Anopheles mosquitoes, the principle vectors of malaria. Here, we report the draft genome sequence of Asaia sp. strain SF2.1. This strain is under development as a platform to deliver antimalarial peptides and proteins to adult female Anopheles mosquitoes.

A persistent problem in modern health care derives from the overwhelming presence of antibiotic-resistant microbes on biomaterials, more specifically, fungal growth on metal-based implants. This study seeks to investigate the antifungal properties of low-level electrochemical treatments delivered using titanium electrodes against Candida albicans. We show that C. albicans can be readily controlled with electrical currents/potentials, reducing the number of viable planktonic cells by 99.7% and biofilm cells by 96.0–99.99%. Additionally, this study explores the ability of the electrochemical treatments to potentiate fluconazole, a clinically used antifungal drug. We have found that electrochemical treatment substantially enhances fluconazole killing activity. While fluconazole alone exhibits a low efficiency against the stationary phase and biofilm cells of C. albicans, complete eradication corresponding to 7-log killing is achieved when the antifungal drug is provided subsequently to the electrochemical treatment. Further mechanistic analyses have revealed that the sequential treatment shows a complex multimodal action, including the disruption of cell wall integrity and permeability, impaired metabolic functions, and enhanced susceptibility to fluconazole, while altering the biofilm structure. Altogether, we have developed and optimized a new therapeutic strategy to sensitize and facilitate the eradication of fluconazole-tolerant microbes from implantable materials. This work is expected to help advance the use of electrochemical approaches in the treatment of infections caused by C. albicans in both nosocomial and clinical cases.

Purpose Corynebacterium spp. are Gram-positive bacteria commonly associated with the ocular surface. Corynebacterium mastitidis was isolated from mouse eyes and was demonstrated to induce a beneficial immune response that can protect the eye from pathogenic infection. Because eye-relevant Corynebacterium spp. are not well described, we generated a C. mast transposon (Tn) mutant library to gain a better understanding of the nature of eye-colonizing bacteria. Methods Tn mutagenesis was performed with a custom Tn5-based transposon that incorporated a promoterless gene for the fluorescent protein mCherry. We screened our library using flow cytometry and enzymatic assays to identify useful mutants that demonstrate the utility of our approach. Results Fluorescence-activated cell sorting (FACS) of mCherry + bacteria allowed us to identify a highly fluorescent mutant that was detectable on the murine ocular surface using microscopy. We also identified a functional knockout that was unable to hydrolyze urea, Urease KO . Although uric acid is an antimicrobial factor produced in tears, Urease KO bacterium maintained an ability to colonize the eye, suggesting that urea hydrolysis is not required for colonization. In vitro and in vivo, both mutants maintained the potential to stimulate protective immunity as compared to wild-type C. mast . Conclusions In sum, we describe a method to genetically modify an eye-colonizing microbe, C. mast. Furthermore, the procedures outlined here will allow for the continued development of genetic tools for modifying ocular Corynebacterium spp., which will lead to a more complete understanding of the interactions between the microbiome and host immunity at the ocular surface.

Recently, our lab discovered that the eye harbors a microbiome that includes Corynebacterium mastitidis, which induces the recruitment of gdT cells and the production of interleukin 17 (IL-17). This local immune response to C. mast can protect the eye from more serious infections, such as Candida albicans and Pseudomonas aeruginosa. Because of C. mast’s ability to stably colonize the ocular surface while inducing a beneficial immune response, we believe that this bacterium has the potential to act as a natural vehicle to deliver therapeutics locally at the ocular surface to alleviate ocular surface disease. Before this can become a reality, a reliable method to genetically modify C. mast is required. Therefore, we took the initial steps toward achieving this goal by genetically modifying C. mast to express genes of interest using transposon mutagenesis. This allowed us to create a mutant library with 1,000+ distinct candidates that expressed the fluorescent protein, mCherry, and kanamycin resistance. Using FACS, we were able to select the brightest C. mast mutants. The brightest four mutants were used to inoculate the eyes of C57BL/6 mice, so that we could: 1) track C. mast in vivo and 2) ensure that the promoters upstream of the genes of interest remain active when C. mast has colonized the ocular mucosa. We were able to image recombinant bacteria directly ex vivo and also confirmed that there was no decrease in bacterial fitness or the protective immune response elicited by C. mast. We concluded from these studies that C. mast can be engineered to express proteins of interest without a reduction in bacterial fitness or protective immunity.