Visualization of Spirochetes by Labeling Membrane Proteins With Fluorescent Biarsenical Dyes.

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

Advanced Human Immunodeficiency Virus Disease in Botswana Following Successful Antiretroviral Therapy Rollout: Incidence of and Temporal Trends in Cryptococcal Meningitis.

Background: Botswana has a well-developed antiretroviral therapy (ART) program that serves as a regional model. With wide ART availability, the burden of advanced human immunodeficiency virus (HIV) and associated opportunistic infections would be expected to decline. We performed a nationwide surveillance study to determine the national incidence of cryptococcal meningitis (CM), and describe characteristics of cases during 2000-2014 and temporal trends at 2 national referral hospitals. Methods: Cerebrospinal fluid data from all 37 laboratories performing meningitis diagnostics in Botswana were collected from the period 2000-2014 to identify cases of CM. Basic demographic and laboratory data were recorded. Complete national data from 2013-2014 were used to calculate national incidence using UNAIDS population estimates. Temporal trends in cases were derived from national referral centers in the period 2004-2014. Results: A total of 5296 episodes of CM were observed in 4702 individuals; 60.6% were male, and median age was 36 years. Overall 2013-2014 incidence was 17.8 (95% confidence interval [CI], 16.6-19.2) cases per 100000 person-years. In the HIV-infected population, incidence was 96.8 (95% CI, 90.0-104.0) cases per 100000 person-years; male predominance was seen across CD4 strata. At national referral hospitals, cases decreased during 2007-2009 but stabilized during 2010-2014. Conclusions: Despite excellent ART coverage in Botswana, there is still a substantial burden of advanced HIV, with 2013-2014 incidence of CM comparable to pre-ART era rates in South Africa. Our findings suggest that a key population of individuals, often men, is developing advanced disease and associated opportunistic infections due to a failure to effectively engage in care, highlighting the need for differentiated care models.

The phenotype of the Cryptococcus-specific CD4+ memory T-cell response is associated with disease severity and outcome in HIV-associated cryptococcal meningitis.

BACKGROUND: Correlates of immune protection in patients with human immunodeficiency virus (HIV)-associated cryptococcal meningitis are poorly defined. A clearer understanding of these immune responses is essential to inform rational development of immunotherapies. METHODS: Cryptococcal-specific peripheral CD4(+) T-cell responses were measured in 44 patients with HIV-associated cryptococcal meningitis at baseline and during follow-up. Responses were assessed following ex vivo cryptococcal mannoprotein stimulation, using 13-color flow-cytometry. The relationships between cryptococcal-specific CD4(+) T-cell responses, clinical parameters at presentation, and outcome were investigated. RESULTS: Cryptococcal-specific CD4(+) T-cell responses were characterized by the production of macrophage inflammatory protein 1α, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α). Conversely, minimal interleukin 4 and interleukin 17 production was detected. Patients surviving to 2 weeks had significantly different functional CD4(+) T-cell responses as compared to those who died. Patients with a response predominantly consisting of IFN-γ or TNF-α production had a 2-week mortality of 0% (0/20), compared with 25% (6/24) in those without this response (P = .025). Such patients also had lower fungal burdens (10 400 vs 390 000 colony-forming units/mL; P < .001), higher cerebrospinal fluid lymphocyte counts (122 vs 8 cells/µL; P < .001), and a trend toward faster rates of clearance of infection. CONCLUSIONS: The phenotype of the peripheral CD4(+) T-cell response to Cryptococcus was associated with disease severity and outcome in HIV-associated cryptococcal meningitis. IFN-γ/TNF-α-predominant responses were associated with survival.

Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa.

BACKGROUND: Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not been experimentally demonstrated. RESULTS: We investigated the contribution of the Bat proteins in the model organism Leptospira biflexa for their potential contributions to growth rate, morphology and protection against oxidative challenges. A genetically engineered mutant strain in which all bat ORFs were deleted did not exhibit altered growth rate or morphology, relative to the wild-type strain. Nor could we demonstrate a protective role for the Bat proteins in coping with various oxidative stresses. Further, pre-exposing L. biflexa to sublethal levels of reactive oxygen species did not appear to induce a general oxidative stress response, in contrast to what has been shown in other bacterial species. Differential proteomic analysis of the wild-type and mutant strains detected changes in the abundance of a single protein only - HtpG, which is encoded by the gene immediately downstream of the bat loci. CONCLUSION: The data presented here do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress as previously proposed. L. biflexa is relatively sensitive to reactive oxygen species such as superoxide and H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe.

A novel predicted bromodomain-related protein affects coordination between meiosis and spermiogenesis in Drosophila and is required for male meiotic cytokinesis.

Temporal coordination of meiosis with spermatid morphogenesis is crucial for successful generation of mature sperm cells. We identified a recessive male sterile Drosophila melanogaster mutant, mitoshell, in which events of spermatid morphogenesis are initiated too early, before meiotic onset. Premature mitochondrial aggregation and fusion lead to an aberrant mitochondrial shell around premeiotic nuclei. Despite successful meiotic karyokinesis, improper mitochondrial localization in mitoshell testes is associated with defective astral central spindles and a lack of contractile rings, leading to meiotic cytokinesis failure. We mapped and cloned the mitoshell gene and found that it encodes a novel protein with a bromodomain-related region. It is conserved in some insect lineages. Bromodomains typically bind to histone acetyl-lysine residues and therefore are often associated with chromatin. The Mitoshell bromodomain-related region is predicted to have an alpha helical structure similar to that of bromodomains, but not all the crucial residues in the ligand-binding loops are conserved. We speculate that Mitoshell may participate in transcriptional regulation of spermatogenesis-specific genes, though perhaps with different ligand specificity compared to traditional bromodomains.