Inside or Out: Characterizing petrobactin use by Bacillus anthracis

Bacillus anthracis is a Gram-positive, spore-forming bacillus and causes the disease anthrax. Anthrax is a deadly infection that begins with phagocytosis of a B. anthracis spore by an antigen presenting cell and ends when bacilli-laden blood from the carcass is exposed to oxygen, which initiates sporulation. Each step in the infection process requires access to nutrients, including iron. Iron is required as a protein co-factor for many different cellular processes but is also tightly regulated within organisms, including both the mammalian host and the bacterium. To gather iron during infection, B. anthracis employs a heme acquisition system as well as two siderophores, petrobactin and bacillibactin. Of these three systems, only petrobactin is required for growth in iron-deplete medium, macrophages, and to cause disease in mouse models of infection. Chapter one describes what is understood about petrobactin including biosynthesis by the asb operon, regulation of biosynthesis, how the ferric-petrobactin complex is imported, and relevance to disease. I also highlight remaining questions such as how petrobactin is exported from the cell and its role in spore biology. In chapter two, I describe my work identifying the petrobactin exporter ApeX. Using a bioinformatics-based protocol to identify putative targets for petrobactin export, I generated single deletion mutants. Laser ablation electrospray ionization mass spectroscopy (LAESI-MS) was adapted to screen for deletion mutants that failed to export petrobactin, enabling identification of ApeX as a petrobactin exporter. An apeX deletion mutant unable to export petrobactin, instead accumulated the molecule within the cell pellet and exported components. These petrobactin components are still able to transport iron and cause disease in a mouse model of inhalational anthrax. I also used LAESI-MS in chapter three to detect petrobactin within B. anthracis spores and explore the role of petrobactin in spore biology. Petrobactin is not required for germination from the spore, but is required for rapid sporulation in the iron-rich ModG sporulation medium. Fluorescent reporters show induction of the asb operon during late stage growth and early sporulation. This phenotype is likely relevant to disease transmission since experiments in defibrinated bovine blood demonstrate that petrobactin is the preferred iron acquisition system during growth in blood and is required for sporulation in aerated blood. Chapter four offers hypotheses and suggestions for how to answer remaining questions not addressed by the research done in this work. I also make hypotheses regarding alternative, non-iron-scavenging functions for petrobactin and discuss the future potential for LAESI-MS as a research tool.PHDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144141/1/akhagan_1.pd

Characterization of FhuA 104/149C: a Double Cysteine FhuA Mutant with Normal Binding and Diminished Transport

Iron is an essential element for most bacteria and is commonly acquired by siderophores, molecules secreted under iron restricted environment to bind ferric iron. Gram negative cells actively uptake these complexes via outer membrane-transport proteins such as FhuA in Escherichia coli. Structural analysis of receptors revealed a conserved β-barrel occluded by an N-terminal plug domain. The cell membrane TonB/ExbB/ExbD complex presumably supplies energy via interaction between the FhuA N-terminal TonB box and the C-terminal domain of TonB. In order to better understand the mechanism of action the FhuA mutant 104/149C, tethering the central β-strands 4 and 6 of the plug domain, was studied and showed severely reduced transport of radio-labeled ferrichrome. In the course of this study, this protein was HPLC purified for structural studies by crystallization and X-ray diffraction. In addition, protein interaction studies were performed with purified TonB-C terminal revealing no impact of the mutation on FhuA-TonB interactions

Factors associated with incomplete childhood immunization in Arbegona district, southern Ethiopia: a case – control study

Abstract Background The prevention of child mortality through immunization is one of the most cost-effective and widely applied public health interventions. In Ethiopia, the Expanded Program on Immunization (EPI) schedule is rarely completed as planned and the full immunization rate is only 24 %. The objective of this study was to identify determinant factors of incomplete childhood immunization in Arbegona district, Sidama zone, southern Ethiopia. Methods A community based unmatched case-control study was undertaken among randomly selected children aged 12 to 23 months and with a total sample size of 548 (183 cases and 365 controls). A multi-stage sampling technique was used to get representative cases and controls. Data was collected using a structured questionnaire and analyzed using SPSS version 16 statistical software. Bivariate and multiple logistic regression analyses were done to identify independent factors for incomplete immunization status of children. Qualitative data were also generated and analyzed using thematic framework. Results The incomplete immunization status of children was significantly associated with young mothers (AOR = 9.54; 95 % CI = 5.03, 18.09), being born second to fourth (AOR = 3.64; 95 % CI = 1.63, 8.14) and being born fifth or later in the family (AOR = 5.27; 95 % CI = 2.20, 12.64) as compared to being born first, a mother’s lack of knowledge about immunization benefits (AOR = 5.51; 95 % CI = 1.52, 19.94) and a mother’s negative perception of vaccine side effects (AOR = 1.92; 95 % CI = 1.01, 3.70). The qualitative finding revealed that the migration of mothers and unavailability of vaccines on appointed immunization dates were the major reasons for partial immunization of children. Conclusion To reduce the number of children with incomplete immunization status, the Arbegona district needs to consider specific planning for mothers with these risk profiles. A focus on strengthening health communication activities to raise immunization awareness and address concerns of vaccine side effects at community level is also needed. This could be achieved through integrating the immunization service to other elements of primary health care.http://deepblue.lib.umich.edu/bitstream/2027.42/116791/1/12889_2015_Article_2678.pd

Mythical numbers and the proceeds of organised crime: estimating mafia proceeds in Italy

Organised crime is a field vulnerable to mythical numbers, i.e. exaggerated estimates lacking empirical support, but acquiring acceptance through repetition. The figures on mafia proceeds in Italy are a striking example of this problem. This study proposes an estimation of mafia proceeds in Italy from nine criminal activities (sexual exploitation of women, illicit firearms trafficking, drug trafficking, counterfeiting, the illicit cigarette trade, illicit gambling, illicit waste disposal, loan sharking, and extortion racketeering) by region and type of mafia (Cosa Nostra, Camorra,\u2018Ndrangheta, Apulian mafias, and other mafias). The results estimate yearly mafia proceeds at approximately \u20ac10.7 bn (0.7% of the Italian GDP), discussing the impact on the regional and national economies and the differences among the types of mafias as to their geographical sources of revenue

Martinez Lake planning area Citizen Advisory Group report

abstract: One of ten citizen reports to be prepared by Long Range Planning Staff in preparation for drafting the Yuma County 2020 Comprehensive Plan. CAG members reviewed the goals, objectives and polices, and the following elements of the Yuma County 2010 Comprehensive Plan: Land Use Element--Martinez Lake Planning Area: Open Spaces & Recreational Resources, Circulation, Environmental, Water Resources, and Safet

Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne

Bacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections

Intestinal calcium and bile salts facilitate germination of <i>Clostridium difficile</i> spores

<div><p><i>Clostridium difficile</i> (<i>C</i>. <i>difficile</i>) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. <i>C</i>. <i>difficile</i> infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. <i>C</i>. <i>difficile</i> spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca²⁺ during <i>C</i>. <i>difficile</i> spore germination and provide direct evidence that intestinal Ca²⁺ coordinates with bile salts to stimulate germination. Endogenous Ca²⁺ (released from within the spore) and a putative AAA+ ATPase, encoded by <i>Cd630_32980</i>, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca²⁺. However, environmental Ca²⁺ replaces glycine as a co-germinant and circumvents the need for endogenous Ca²⁺ fluxes. <i>Cd630_32980</i> is dispensable for colonization in a murine model of <i>C</i>. <i>difficile</i> infection and <i>ex vivo</i> germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca²⁺ present within the gastrointestinal tract plays a critical role in <i>C</i>. <i>difficile</i> germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (<i>e</i>.<i>g</i>., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.</p></div

Exogenous calcium induces <i>C</i>. <i>difficile</i> germination in concert with taurocholate.

<p>Cd630, VPI 10463, or R20291 spores were incubated with the indicated combinations of 0.2% Tc, 60 mM Ca-DPA, 50 mM glycine, 60 mM CaCl₂, or 60 mM DPA (A-G, I, J). Activation of SleC was assessed by western blot analyses. Cd630 spores were incubated for 15 minutes at 37°C with the indicated combinations of 1% Tc, 50 mM glycine, 60 mM CaCl₂, 60 mM DPA, or 60 mM Ca-DPA. Spores were subsequently lysed and assayed for levels of pro-SleC and SleC (K). <i>Bacillus anthracis</i> strain Sterne 34F₂ spores were incubated with the indicated combinations of either 60 mM CaCl₂, 60 mM DPA, or 60 mM CaDPA (H). Germination was measured either by tracking the loss of OD₆₀₀ over time (A-H), measuring loss of heat resistance at 37°C after 1 hour (I), or measuring release of DPA at 37°C after 1 hour (J). Germination assays were performed in triplicate. Germination assays and western blots are representative of three independent spore preps. Error bars are mean plus or minus SD. Statistical analysis was performed using one-way ANOVA. (****) p<0.0001.</p

<i>Cd630_32980</i> is required for Tc-Gly induced germination.

<p>WT and Δ<i>32980</i> spores were incubated with 0.2% Tc and the indicated concentrations of glycine (a-b) or CaCl₂ (c-d) with germination was measured by loss of OD₆₀₀ at 37°C over the course of an hour. Western blot assessing SleC activation from WT and Δ<i>32980</i> spores incubated at 37°C for 15 minutes with 1% Tc and the indicated concentrations of glycine (e) or CaCl₂ (f). %pro-SleC and % SleC relative densities were calculated for each lane using ImageJ by determining the ratio of each band to the total density for the two bands combined. Blots are representative of 3 experiments. Germination assays were performed in triplicate and are representative of 3 independent spore preps. Error bars are mean plus or minus SD.</p