15 research outputs found
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 24Â %. 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 23Â 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
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 Ca<sup>2+</sup> during <i>C</i>. <i>difficile</i> spore germination and provide direct evidence that intestinal Ca<sup>2+</sup> coordinates with bile salts to stimulate germination. Endogenous Ca<sup>2+</sup> (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 Ca<sup>2+</sup>. However, environmental Ca<sup>2+</sup> replaces glycine as a co-germinant and circumvents the need for endogenous Ca<sup>2+</sup> 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 Ca<sup>2+</sup> 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 CaCl<sub>2</sub>, 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 CaCl<sub>2</sub>, 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 34F<sub>2</sub> spores were incubated with the indicated combinations of either 60 mM CaCl<sub>2</sub>, 60 mM DPA, or 60 mM CaDPA (H). Germination was measured either by tracking the loss of OD<sub>600</sub> 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 CaCl<sub>2</sub> (c-d) with germination was measured by loss of OD<sub>600</sub> 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 CaCl<sub>2</sub> (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
Proposed model for the role of calcium in <i>C</i>. <i>difficile</i> germination.
<p>Tc binds to CspC (A) facilitating movement of glycine or calcium through the spore coat and outer membrane (B). Glycine then interacts with an unknown receptor (C) inducing the release of Ca<sup>2+</sup> from the spore core (D). Ca<sup>2+</sup> from the environment or the spore core activates CspB (E), which processes pro-SleC (F) subsequently initiating cortex hydrolysis (G). This leads to full core rehydration (H), complete release of DPA (I) and spore outgrowth.</p
Tc-CaCl<sub>2</sub> induced germination occurs through the same pathway as Tc-Gly.
<p>Cd630, Δ<i>gerS</i>, Δ<i>cspB</i>, Δ<i>cspC</i>, and Δ<i>sleC</i> spores were incubated with 0.2% Tc and with (+) or without (-) the indicated co-cogerminant: 50 mM glycine (a) or 60 mM CaCl<sub>2</sub> (b). Germination was measured by loss of OD<sub>600</sub> at 37°C over the course of one hour. (a-b). Western blot of SleC from WT, Δ<i>gerS</i>, Δ<i>cspB</i>, or Δ<i>cspC</i> spores incubated at 37°C for 15 minutes with 1% Tc and the indicated concentrations of glycine (c) or CaCl<sub>2</sub> (d). Germination assays were performed in triplicate. Germination assays and western blots are representative of 3 independent spore preps. Error bars are mean plus or minus SD.</p
Calcium from within the spore is essential for germination in response to Tc-Gly.
<p>Cd630 spores were incubated with 0.2% Tc, 50mM glycine and different concentrations of EGTA (A). Cd630 spores were incubated with the indicated combinations of 0.2% Tc, 50 mM glycine, 1 mM CaCl<sub>2</sub>, and 50 μM EGTA (B). EGTA (1mM) pretreated Cd630 spores and non-treated Cd630 spores were incubated with the indicated combinations of chelex-treated germinants, Tc (0.2%) and glycine (50mM) (C). Activation of SleC was assessed by western blot. Cd630 spores were incubated 37°C for 15 minutes with 1% Tc and the indicated combinations of 50mM glycine and 1mM EGTA (D). Germination was tracked by loss of OD at 37°C over the course of one hour. Germination assays were performed in triplicate. Germination assays and western blots are representative of 3 independent spore preps. Error bars are mean plus or minus SD.</p
Tc-Gly induced germination is dispensable for <i>in vivo</i> germination of <i>C</i>. <i>difficile</i> spores.
<p>Cefoperazone-treated C57BL/6 mice (n = 8) were infected with 1500 spores of WT or Δ<i>32980</i> by oral gavage. Colonization levels were assessed daily and are presented in total CFU per gram of feces. (a) Multiple t tests were performed and p = 0.23 for each time point tested (a). Ileal contents were collected from Cefoperazone treated C57BL/6 mice (n = 3) and <i>ex vivo</i> germination assays were performed. 1x10<sup>3</sup> spores of Cd630 or Δ<i>32980</i> were incubated for one hour at 37°C in ileal contents, calcium depleted ileal contents or calcium depleted ileal contents treated with 15 mM CaCl<sub>2</sub>. Samples were then incubated at 65°C for 20 min and then plated on BHIS-Tc plates. Data are presented as % loss of Heat Resistance (b). Free calcium levels of ileal contents, calcium depleted ileal contents or calcium depleted ileal contents treated with 15 mM CaCl<sub>2</sub> were measured using a calcium colorimetric assay. Levels of calcium (mM) were determined using a standard curve (c). Assays were performed in triplicate using ileal contents from three mice. Error bars are mean plus or minus SD. Statistical significance was calculated using Two-way ANOVA. (*) p<0.05 (****) p<0.0001.</p