Cryptococcus neoformans activates RhoGTPase proteins followed by protein kinase C, focal adhesion kinase, and ezrin to promote traversal across the blood-brain barrier.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.

Natural niche for organohalide-respiring Chloroflexi.

The phylum Chloroflexi contains several isolated bacteria that have been found to respire a diverse array of halogenated anthropogenic chemicals. The distribution and role of these Chloroflexi in uncontaminated terrestrial environments, where abundant natural organohalogens could function as potential electron acceptors, have not been studied. Soil samples (116 total, including 6 sectioned cores) from a range of uncontaminated sites were analyzed for the number of Dehalococcoides-like Chloroflexi 16S rRNA genes present. Dehalococcoides-like Chloroflexi populations were detected in all but 13 samples. The concentrations of organochlorine ([organochlorine]), inorganic chloride, and total organic carbon (TOC) were obtained for 67 soil core sections. The number of Dehalococcoides-like Chloroflexi 16S rRNA genes positively correlated with [organochlorine]/TOC while the number of Bacteria 16S rRNA genes did not. Dehalococcoides-like Chloroflexi were also observed to increase in number with a concomitant accumulation of chloride when cultured with an enzymatically produced mixture of organochlorines. This research provides evidence that organohalide-respiring Chloroflexi are widely distributed as part of uncontaminated terrestrial ecosystems, they are correlated with the fraction of TOC present as organochlorines, and they increase in abundance while dechlorinating organochlorines. These findings suggest that organohalide-respiring Chloroflexi may play an integral role in the biogeochemical chlorine cycle.

Contrasting patterns of genome-level diversity across distinct co-occurring bacterial populations.

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single-cell genomes and a 5-year metagenomic time series. The single-cell genomes grouped into 15 monophyletic clusters (termed "tribes") that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI Actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.

Erratum for Linz et al., "Bacterial Community Composition and Dynamics Spanning Five Years in Freshwater Bog Lakes".

[This corrects the article DOI: 10.1128/mSphere.00169-17.].

Bacterial Community Composition and Dynamics Spanning Five Years in Freshwater Bog Lakes.

Bacteria play a key role in freshwater biogeochemical cycling, but long-term trends in freshwater bacterial community composition and dynamics are not yet well characterized. We used a multiyear time series of 16S rRNA gene amplicon sequencing data from eight bog lakes to census the freshwater bacterial community and observe annual and seasonal trends in abundance. The sites that we studied encompassed a range of water column mixing frequencies, which we hypothesized would be associated with trends in alpha and beta diversity. Each lake and layer contained a distinct bacterial community, with distinct levels of richness and indicator taxa that likely reflected the environmental conditions of each lake type sampled, including Actinobacteria in polymictic lakes (i.e., lakes with multiple mixing events per year), Methylophilales in dimictic lakes (lakes with two mixing events per year, usually in spring and fall), and "Candidatus Omnitrophica" in meromictic lakes (lakes with no recorded mixing events). The community present during each year at each site was also surprisingly unique. Despite unexpected interannual variability in community composition, we detected a core community of taxa found in all lakes and layers, including Actinobacteria tribe acI-B2 and Betaprotobacteria lineage PnecC. Although trends in abundance did not repeat annually, each freshwater lineage within the communities had a consistent lifestyle, defined by persistence, abundance, and variability. The results of our analysis emphasize the importance of long-term multisite observations, as analyzing only a single year of data or one lake would not have allowed us to describe the dynamics and composition of these freshwater bacterial communities to the extent presented here. IMPORTANCE Lakes are excellent systems for investigating bacterial community dynamics because they have clear boundaries and strong environmental gradients. The results of our research demonstrate that bacterial community composition varies by year, a finding which likely applies to other ecosystems and has implications for study design and interpretation. Understanding the drivers and controls of bacterial communities on long time scales would improve both our knowledge of fundamental properties of bacterial communities and our ability to predict community states. In this specific ecosystem, bog lakes play a disproportionately large role in global carbon cycling, and the information presented here may ultimately help refine carbon budgets for these lakes. Finally, all data and code in this study are publicly available. We hope that this will serve as a resource for anyone seeking to answer their own microbial ecology questions using a multiyear time series.

Abundance and diversity of organohalide-respiring bacteria in lake sediments across a geographical sulfur gradient.

Across the U.S. Upper Midwest, a natural geographical sulfate gradient exists in lakes. Sediment grab samples and cores were taken to explore whether this sulfur gradient impacted organohalide-respiring Chloroflexi in lake sediments. Putative organohalide-respiring Chloroflexi were detected in 67 of 68 samples by quantitative polymerase chain reaction. Their quantities ranged from 3.5 × 10(4) to 8.4 × 10(10) copies 16S rRNA genes g(-1) dry sediment and increased in number from west to east, whereas lake sulfate concentrations decreased along this west-to-east transect. A terminal restriction fragment length polymorphism (TRFLP) method was used to corroborate this inverse relationship, with sediment samples from lower sulfate lakes containing both a higher number of terminal restriction fragments (TRFs) belonging to the organohalide-respiring Dehalococcoidetes, and a greater percentage of the TRFLP amplification made up by Dehalococcoidetes members. Statistical analyses showed that dissolved sulfur in the porewater, measured as sulfate after oxidation, appeared to have a negative impact on the total number of putative organohalide-respiring Chloroflexi, the number of Dehalococcoidetes TRFs, and the percentage of the TRFLP amplification made up by Dehalococcoidetes. These findings point to dissolved sulfur, presumably present as reduced sulfur species, as a potentially controlling factor in the natural cycling of chlorine, and perhaps as a result, the natural cycling of some carbon as well.