Correction: Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

[This corrects the article DOI: 10.1371/journal.ppat.1007470.].

Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco_Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco_Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.

Trypanosome infection establishment in the tsetse fly gut is influenced by microbiome-regulated host immune barriers.

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host's physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse's resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm(Apo)) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm(Apo) adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm(Apo) flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse's PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse's larval microbiome and the integrity of the emerging adult PM gut immune barrier.

OmpA-mediated biofilm formation is essential for the commensal bacterium Sodalis glossinidius to colonize the tsetse fly gut.

Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossina spp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in the tsetse gut and that this process is influenced by the Sodalis outer membrane protein A (OmpA). Mutant Sodalis strains that do not produce OmpA (Sodalis ΔOmpA mutants) fail to form biofilms in vitro and are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, Sodalis ΔOmpA mutant cells are exposed to and eliminated by tsetse's innate immune system, suggesting that biofilms help Sodalis evade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promote Sodalis colonization of the tsetse gut may enhance the development of novel disease control strategies.

Metagenomic analysis of the medicinal leech gut microbiota.

There are trillions of microbes found throughout the human body and they exceed the number of eukaryotic cells by 10-fold. Metagenomic studies have revealed that the majority of these microbes are found within the gut, playing an important role in the host's digestion and nutrition. The complexity of the animal digestive tract, unculturable microbes, and the lack of genetic tools for most culturable microbes make it challenging to explore the nature of these microbial interactions within this niche. The medicinal leech, Hirudo verbana, has been shown to be a useful tool in overcoming these challenges, due to the simplicity of the microbiome and the availability of genetic tools for one of the two dominant gut symbionts, Aeromonas veronii. In this study, we utilize 16S rRNA gene pyrosequencing to further explore the microbial composition of the leech digestive tract, confirming the dominance of two taxa, the Rikenella-like bacterium and A. veronii. The deep sequencing approach revealed the presence of additional members of the microbial community that suggests the presence of a moderately complex microbial community with a richness of 36 taxa. The presence of a Proteus strain as a newly identified resident in the leech crop was confirmed using fluorescence in situ hybridization (FISH). The metagenome of this community was also pyrosequenced and the contigs were binned into the following taxonomic groups: Rikenella-like (3.1 MB), Aeromonas (4.5 MB), Proteus (2.9 MB), Clostridium (1.8 MB), Eryspelothrix (0.96 MB), Desulfovibrio (0.14 MB), and Fusobacterium (0.27 MB). Functional analyses on the leech gut symbionts were explored using the metagenomic data and MG-RAST. A comparison of the COG and KEGG categories of the leech gut metagenome to that of other animal digestive-tract microbiomes revealed that the leech digestive tract had a similar metabolic potential to the human digestive tract, supporting the usefulness of this system as a model for studying digestive-tract microbiomes. This study lays the foundation for more detailed metatranscriptomic studies and the investigation of symbiont population dynamics.