Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling inducing anemia.

Parasitic unicellular eukaryotes use extracellular vesicles (EVs) as vehicles for intercellular communication and host manipulation. By using various mechanisms to generate EVs and by transferring a wide range of molecules through EVs, pathogenic protozoans are able to establish infective niches, modulate the immune system of the host and cause disease. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this Progress article, we explore recent insights into the biology of EVs from human infectious protozoan parasites, including Trichomonas vaginalis, Plasmodium spp. and kinetoplastids, such as Trypanosoma spp. and Leishmania spp.

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other’s TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically up-regulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.

This study examined seasonal differences in microbial community structure in the sediment of three streams in North Carolina's Neuse River Basin. Microbes that reside in sediment are at the base of the food chain and have a profound influence on the health of freshwater stream environments. Terminal-Restriction Fragment Length Polymorphism (T-RFLP), molecular fingerprint analysis of 16S rRNA genes was used to examine the diversity of bacterial species in stream sediment. Sediment was sampled in both wet and dry seasons from an agricultural (Bear), mixed urban (Crabtree) and forested (Marks) Creek, and the microbiota examined. Gamma, Alpha and Beta proteobacteria were prevalent species of microbial taxa represented among all sites. Actinobacteria was the next most prevalent species observed, with greater occurrence in dry compared to the wet season. Discernable clustering was observed of Marks and Bear Creek samples collected during the wetter period (September-April), which corresponded with a period of higher OPEN ACCESSDiversity 2014, 6 19 precipitation and cooler surface water temperatures. Although not statistically significant, microbial community structure appeared different between season (ANOSIM, R = 0.60; p < 0.10). Principal components analysis confirmed this pattern and showed that the bacterial groups were separated by wet and dry seasonal periods. These results suggest seasonal differences among the microbial community structure in sediment of freshwater streams and that these communities may respond to changes in precipitation during wetter periods.

␣-Ketoglutarate decarboxylase (␣-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, ␣-KDE1 was expressed in BF T. brucei and RNA interference knockdown of ␣-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of ␣-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of ␣-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of ␣-KDE1 contain overlapping mitochondrion-and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that ␣-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.

Trypanosomes possess a unique mitochondrial genome called the kinetoplast DNA (kDNA). Many kDNA genes encode premRNAs that must undergo guide RNA-directed editing. In addition, alternative mRNA editing gives rise to diverse mRNAs and several kDNA genes encode open reading frames of unknown function. To better understand the mechanism of RNA editing and the function of mitochondrial RNAs in trypanosomes, we have developed a reverse genetic approach using artificial sitespecific RNA endonucleases (ASREs) to directly silence kDNA-encoded genes. The RNA-binding domain of an ASRE can be programmed to recognize unique 8-nucleotide sequences, allowing the design of ASREs to cleave any target RNA. Utilizing an ASRE containing a mitochondrial localization signal, we targeted the extensively edited mitochondrial mRNA for the subunit A6 of the F 0 F 1 ATP synthase (A6) in the procyclic stage of Trypanosoma brucei. This developmental stage, found in the midgut of the insect vector, relies on mitochondrial oxidative phosphorylation for ATP production with A6 forming the critical proton half channel across the inner mitochondrial membrane. Expression of an A6-targeted ASRE in procyclic trypanosomes resulted in a 50% reduction in A6 mRNA levels after 24 h, a time-dependent decrease in mitochondrial membrane potential (ΔΨm), and growth arrest. Expression of the A6-ASRE, lacking the mitochondrial localization signal, showed no significant growth defect. The development of the A6-ASRE allowed the first in vivo functional analysis of an edited mitochondrial mRNA in T. brucei and provides a critical new tool to study mitochondrial RNA biology in trypanosomes.

Summary The spirochaete (Borrelia burgdorferi) associated with Lyme disease was detected in questing ticks and rodents during a period of 18 years, 1991–2009, at five locations on the Outer Banks of North Carolina. The black-legged tick (Ixodes scapularis) was collected at varied intervals between 1991 and 2009 and examined for B. burgdorferi. The white-footed mouse (Peromyscus leucopus), house mouse (Mus musculus) marsh rice rat (Oryzomys palustris), marsh rabbit (Sylvilagus palustris), eastern cottontail (Sylvilagus floridanus) and six-lined racerunner (Cnemidophorus sexlineatus) were live-trapped, and their tissues cultured to isolate spirochaetes. Borrelia burgdorferi isolates were obtained from questing adult I. scapularis and engorged I. scapularis removed from P. leucopus, O. palustris and S. floridanus. The prevalence of B. burgdorferi infection was variable at different times and sites ranging from 7 to 14% of examined questing I. scapularis. Mitochondrial (16S) rRNA gene phylogenetic analysis from 65 adult I. scapularis identified 12 haplotypes in two major clades. Nine haplotypes were associated with northern/Midwestern I. scapularis populations and three with southern I. scapularis populations. Sixteen isolates obtained from tick hosts in 2005 were confirmed to be B. burgdorferi by amplifying and sequencing of 16S rRNA and 5S–23S intergenic spacer fragments. The sequences had 98–99% identity to B. burgdorferi sensu stricto strains B31, JD1 and M11p. Taken together, these studies indicate that B. burgdorferi sensu stricto is endemic in questing I. scapularis and mammalian tick hosts on the Outer Banks of North Carolina.

Exposure to ultraviolet light leads to a cell-wide DNA damage response that includes a global reduction in transcription. Williamson et al., identify a protein involved in this process as well as a noncoding RNA produced by alternative processing of RNA transcribed from the same gene that promotes recovery from the repressed state.