Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.

Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

Bacteria localization and chorion thinning among preterm premature rupture of membranes.

OBJECTIVE: Bacterial colonization of the fetal membranes and its role in pathogenesis of membrane rupture is poorly understood. Prior retrospective work revealed chorion layer thinning in preterm premature rupture of membranes (PPROM) subjects. Our objective was to prospectively examine fetal membrane chorion thinning and to correlate to bacterial presence in PPROM, preterm, and term subjects. STUDY DESIGN: Paired membrane samples (membrane rupture and membrane distant) were prospectively collected from: PPROM = 14, preterm labor (PTL = 8), preterm no labor (PTNL = 8), term labor (TL = 10), and term no labor (TNL = 8), subjects. Sections were probed with cytokeratin to identify fetal trophoblast layer of the chorion using immunohistochemistry. Fluorescence in situ hybridization was performed using broad range 16 s ribosomal RNA probe. Images were evaluated, chorion and choriodecidua were measured, and bacterial fluorescence scored. Chorion thinning and bacterial presence were compared among and between groups using Student's t-test, linear mixed effect model, and Poisson regression model (SAS Cary, NC). RESULTS: In all groups, the fetal chorion cellular layer was thinner at rupture compared to distant site (147.2 vs. 253.7 µm, p<0.0001). Further, chorion thinning was greatest among PPROM subjects compared to all other groups combined, regardless of site sampled [PPROM(114.9) vs. PTL(246.0) vs. PTNL(200.8) vs. TL(217.9) vs. TNL(246.5)]. Bacteria counts were highest among PPROM subjects compared to all other groups regardless of site sampled or histologic infection [PPROM(31) vs. PTL(9) vs. PTNL(7) vs. TL(7) vs. TNL(6)]. Among all subjects at both sites, bacterial counts were inversely correlated with chorion thinning, even excluding histologic chorioamnionitis (p<0.0001 and p = 0.05). CONCLUSIONS: Fetal chorion was uniformly thinner at rupture site compared to distant sites. In PPROM fetal chorion, we demonstrated pronounced global thinning. Although cause or consequence is uncertain, bacterial presence is greatest and inversely correlated with chorion thinning among PPROM subjects.

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

Programming stress-induced altruistic death in engineered bacteria.

Programmed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is 'altruistic': the killing of some cells can benefit the survivors through release of 'public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, we determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. We further predicted that altruistic death could generate the 'Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, we experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. Our findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment.

Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

A noisy linear map underlies oscillations in cell size and gene expression in bacteria.

During bacterial growth, a cell approximately doubles in size before division, after which it splits into two daughter cells. This process is subjected to the inherent perturbations of cellular noise and thus requires regulation for cell-size homeostasis. The mechanisms underlying the control and dynamics of cell size remain poorly understood owing to the difficulty in sizing individual bacteria over long periods of time in a high-throughput manner. Here we measure and analyse long-term, single-cell growth and division across different Escherichia coli strains and growth conditions. We show that a subset of cells in a population exhibit transient oscillations in cell size with periods that stretch across several (more than ten) generations. Our analysis reveals that a simple law governing cell-size control-a noisy linear map-explains the origins of these cell-size oscillations across all strains. This noisy linear map implements a negative feedback on cell-size control: a cell with a larger initial size tends to divide earlier, whereas one with a smaller initial size tends to divide later. Combining simulations of cell growth and division with experimental data, we demonstrate that this noisy linear map generates transient oscillations, not just in cell size, but also in constitutive gene expression. Our work provides new insights into the dynamics of bacterial cell-size regulation with implications for the physiological processes involved.

Variability in phenotype induced by the podocin variant R229Q plus a single pathogenic mutation.

BACKGROUND: Mutations in podocin (NPHS2) are the most common cause of childhood onset autosomal recessive steroid-resistant nephrotic syndrome (SRNS). The disease is characterized by early-onset proteinuria, resistance to immunosuppressive therapy and rapid progression to end-stage renal disease. Compound heterozygous changes involving the podocin variant R229Q combined with another pathogenic mutation have been associated with a mild phenotype with disease onset often in adulthood. METHODS: We screened 19 families with early-onset SRNS for mutations in NPHS2 and WT1 and identified four disease-causing mutations (three in NPHS2 and one in WT1) prior to planned whole-exome sequencing. RESULTS: We describe two families with three individuals presenting in childhood who are compound heterozygous for R229Q and one other pathogenic NPHS2 mutation, either L327F or A297V. One child presented at age 4 years (A297V plus R229Q) and the other two at age 13 (L327F plus R229Q), one with steadily deteriorating renal function. CONCLUSIONS: These cases highlight the phenotypic variability associated with the NPHS2 R229Q variant plus pathogenic mutation. Individuals may present with early aggressive disease.