Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response

Drop impact on superheated surfaces

At impact of a liquid droplet on a smooth surface heated above the liquid's boiling point, the droplet either immediately boils when it contacts the surfaces (``contact boiling''), or without any surface contact forms a Leidenfrost vapor layer towards the hot surface and bounces back (``gentle film boiling''), or both forms the Leidenfrost layer and ejects tiny droplets upward (``spraying film boiling''). We experimentally determine conditions under which impact behaviors in each regime can be realized. We show that the dimensionless maximum spreading $\gamma$ of impacting droplets on the heated surfaces in both gentle and spraying film boiling regimes shows a universal scaling with the Weber number \We (\gamma\sim\We^{2/5}) -- regardless of surface temperature and of liquid properties -- which is much steeper than for the impact on non-heated (hydrophilic or hydrophobic) surfaces (\gamma\sim\We^{1/4}). We also intereferometrically measure the vapor thickness under the droplet

Marangoni shocks in unobstructed soap-film flows

It is widely thought that in steady, gravity-driven, unobstructed soap-film flows, the velocity increases monotonically downstream. Here we show experimentally that the velocity increases, peaks, drops abruptly, then lessens gradually downstream. We argue theoretically and verify experimentally that the abrupt drop in velocity corresponds to a Marangoni shock, a type of shock related to the elasticity of the film. Marangoni shocks induce locally intense turbulent fluctuations and may help elucidate the mechanisms that produce two-dimensional turbulence away from boundaries.Comment: 4 pages, 5 figures, published in PR

Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host

A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.IMPORTANCE Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi Our results suggest that H. ducreyi survives in an abscess by utilizing l-ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection

Decoding the complexities of human malaria through systems immunology

The complexity of the Plasmodium parasite and its life cycle poses a challenge to our understanding of the host immune response against malaria. Studying human immune responses during natural and experimental Plasmodium infections can enhance our understanding of malaria-protective immunity and inform the design of disease-modifying adjunctive therapies and next-generation malaria vaccines. Systems immunology can complement conventional approaches to facilitate our understanding of the complex immune response to the highly dynamic malaria parasite. In this review, recent studies that used systems-based approaches to evaluate human immune responses during natural and experimental Plasmodium falciparum and Plasmodium vivax infections as well as during immunization with candidate malaria vaccines are summarized and related to each other. The potential for next-generation technologies to address the current limitations of systems-based studies of human malaria are discussed

Extraction of active, contaminant degrading enzymes from soil

Soil microorganisms play critical roles in the degradation of micro-and nano-pollutants, and the corresponding proteins and enzymes play roles in pollutant recognition, transportation, and degradation. Our ability to study these pathways from soil samples is often complicated by the complex processes involved in extracting proteins from soil matrices. This study aimed to develop a new protein soil extraction protocol that yielded active, intracellular enzymes from the perchlorate degradation pathway, particularly perchlorate reductase. An indirect method, which focused on first separating the cells from the soil matrix, followed by cell lysis and enzyme extraction, was evaluated. The optimized indirect method achieved a final extraction efficiency of the active enzyme and total protein of 15.7 % and 3.3 %, respectively. The final step of separating enzymes from residual soil components resulted in the highest activity and protein losses of 67.7 % ± 14.8 % and 91.8 % ± 1.8 %, respectively. Five buffers, each at different concentrations (0.01 M, 0.05 M, and 0.1 M), were tested to enhance enzyme extraction efficiency. The best extractant requires careful consideration between the highest activity and the quality of the recovered enzymes. Coextraction of humic substances could be minimized by using 0.1 M as compared to 0.01 M and 0.05 M of sodium pyrophosphate; however, this resulted in less recovered activity compared to lower extractant concentrations

Whole-blood transcriptomic signatures induced during immunization by chloroquine prophylaxis and Plasmodium falciparum sporozoites

A highly effective vaccine that confers sterile protection to malaria is urgently needed. Immunization under chemoprophylaxis with sporozoites (CPS) consistently confers high levels of protection in the Controlled Human Malaria infection (CHMI) model. To provide a broad, unbiased assessment of the composition and kinetics of direct ex vivo human immune responses to CPS, we profiled whole-blood transcriptomes by RNA-seq before and during CPS immunization and following CHMI challenge. Differential expression of genes enriched in modules related to T cells, NK cells, protein synthesis, and mitochondrial processes were detected in fully protected individuals four weeks after the first immunization. Non-protected individuals demonstrated transcriptomic changes after the third immunization and the day of treatment, with upregulation of interferon and innate inflammatory genes and downregulation of B-cell signatures. Protected individuals demonstrated more significant interactions between blood transcription modules compared to non-protected individuals several weeks after the second and third immunizations. These data provide insight into the molecular and cellular basis of CPS-induced immune protection from P. falciparum infection

Roles of GRK and PDE4 Activities in the Regulation of β2 Adrenergic Signaling

An important focus in cell biology is understanding how different feedback mechanisms regulate G protein–coupled receptor systems. Toward this end we investigated the regulation of endogenous β2 adrenergic receptors (β2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 μM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 μM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 μM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 μM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of β2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of β2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system