Retos actuales de la farmacia

Retos actuales de la farmacia es un proyecto que está coordinado por Leobargo Manuel Gómez Oliván y un equipo de investigadores que forman parte del claustro de la Facultad de Química en el área de posgrado, ellos han incentivado el espíritu investigador y científico de los estudiantes adscritos al programa para adentrarse en el ámbito farmacéutico. Los capítulos que conforman esta edición son el reflejo de la actividad académica desarrollada en este posgrado en las diferentes áreas de acentuación que lo conforman: farmacia molecular, farmacia social y tecnología farmacéutica

Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis.

Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis

Colonization of <i>C</i>. <i>difficile</i> spores of wild-type, <i>cdeC</i> and <i>cdeM</i> strains in a murine model of infection.

<p>Mice were treated with antibiotics for 3 days, followed by intraperitoneal injection of clindamycin one day prior to infection via oral gavage with 2x10⁷ spores of a 1:1 mixture of: <b>(A, C)</b> <i>C</i>. <i>difficile</i> 630<i>erm</i> wild-type and <i>cdeC</i> mutant spores; or <b>(B, D)</b> <i>C</i>. <i>difficile</i> 630<i>erm</i> wild-type and <i>cdeM</i> mutant spores. Fecal shedding of 630<i>erm</i> wild-type and <i>cdeC</i> or <i>cdeM</i> mutants was quantified as described in the Method section. <b>(A, C)</b> Persistence of 630<i>erm</i> wild-type and <i>cdeC</i> mutant <b>(A)</b> or <i>cdeC</i> mutant <b>(C)</b> in fecal samples. <b>(B, D)</b> Competitive index (CI) course of 630<i>erm</i> wild-type and <i>cdeM</i> mutant <b>(B)</b> or wild-type and <i>cdeM</i> mutant <b>(D)</b> monitored over 8 days. Open circles indicate CI values from individual mice and the red horizontal bars indicate the geometric means. Mann-Whitney test was used to determine statistical differences between strains. Asterisks (*) indicate statistical difference with a <i>P</i>-value < 0.05. LOD, limit of detection is indicated by the dotted line.</p

Absence of CdeC and CdeM renders <i>C</i>. <i>difficile</i> spores susceptible to ethanol, heat and macrophages.

<p><b>(A)</b> Ethanol 50% resistance of wild-type (white bar), <i>cdeC</i> (gray bar) and <i>cdeM</i> (black bar) spores. <b>(B)</b> Heat resistance of <i>C</i>. <i>difficile</i> wild-type (gray white), <i>cdeC</i> (gray bars), and <i>cdeM</i> (black bars) spores was measured by heat treating aliquots at 75°C for various times, and survivors were enumerated as described in the Material and Method section. <b>(C)</b> Equal amounts of spores derived from <i>C</i>. <i>difficile</i> strains 630<i>erm</i> (wt), <i>cdeC</i>, <i>cdeM</i>, <i>cdeC/cdeC</i> and <i>cdeM/cdeM</i> were boiled 60 min, and the amount of DPA was quantified based on Tb³⁺. The data shown represent the average results from three independent experiments, and the error bars represent standard error from the means. n.s., indicates no significant difference relative to wild-type. <b>(D)</b> Resistance of Raw 264.7 macrophages was determined by infecting at a MOI of 10 with <i>C</i>. <i>difficile</i> 630<i>erm</i> wild-type, <i>cdeC</i> and <i>cdeM</i> after 0,5, 24 and 48 of incubation at 37°C. Asterisks (*) denote statistical difference at <i>P</i> < 0.01 respect to wild-type.</p

Protein profile and immunoreactive bands of <i>C</i>. <i>difficile</i> spores in the absence of CdeC and CdeM.

<p><b>(A)</b> Coat and exosporium extracts of 4x10⁷ spores of each strain were electrophoresed and stained with Coomassie brilliant blue. Black arrows highlight the major protein bands. <b>(B)</b>, Densitometry analysis of the relative major protein bands in an SDS-PAGE gel. <b>(C)</b> Western blot of spore coat/exosporium fractions of wild-type, <i>cdeC</i> and <i>cdeM</i> spores blotted with rabbit anti-SpoIVA. Densitometric analysis of SpoIVA-immunoreactive bands were done with Image-J. <b>(D)</b>, Western blot analysis of spore coat/exosporium fractions of wild-type, <i>cdeC</i> and <i>cdeM</i> spores blotted with goat antiserum raised against <i>C</i>. <i>difficile</i> 630<i>erm</i> spore. Black arrows highlight the immunoreactive bands. Densitometry analysis of the major imunoreactive bands was determined with ImageJ, and the results are expressed as relative to those determined in wild-type spores. The SDS-PAGE and Western blots are a representative experiment. Data of densitometric analysis represent the mean of three representative experiments and error bars are standard error of the mean. Asterisks denote statistical difference at (*) <i>P</i> < 0.01, (**) <i>P</i> < 0.05 and (***) <i>P</i> < 0.001 respect to wild-type.</p

Effect of CdeC and CdeM in the initiation and recurrence of the disease in a mouse model of infection.

<p><b>(A)</b> Overview of the experimental design for CDI-R mouse model. Cefoperazone treated C57BL/6 mice were infected with 3x10⁷ <i>C</i>. <i>difficile</i> strain 630<i>erm</i> wild-type (n = 6), <i>cdeC</i> (n = 6) or <i>cdeM</i> (n = 5). <b>(B)</b> time to diarrhea during the first episode; <b>(C)</b> animals, were treated with vancomicyn for 5 days to induce CDI-R and animals were monitored during CDI-R for time to diarrhea during recurrence; <b>(D)</b> cecum content cytotoxicity; <b>(E)</b> <i>C</i>. <i>difficile</i> spores in cecum tissue. Error bars are standard error of the mean. (Kruskal Wallis, post Dunnett test, P < 0.05); n.s, is no significance.</p

Schematic representation of CdeC and CdeM proteins and their conservation in <i>C</i>. <i>difficile</i> and other Peptostreptococcaceae family members.

<p><b>(A)</b><i>cdeC</i> is found downstream of <i>CD1066</i>, which encodes a putative protein of unknown function protein and upstream of <i>CD1068</i> which is an antisense encoding ORF of a putative protein involved in polysaccharide biosynthesis, and expressed during sporulation protein encoded in the antisense complementary sequence; all three are monocistronic genes. Notably, a putative σ^K-regulated promoter is located immediately upstream of <i>cdeC</i>, whose position was mapped by RNA-Seq [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref028" target="_blank">28</a>] (shown in the scheme). By contrast, <i>CD1066</i> and <i>CD1068</i> have putative σ^E-regulated promoter immediately upstream of their ORFs [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref028" target="_blank">28</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref030" target="_blank">30</a>]. By contrast, <i>cdeM</i> is found downstream of <i>CD1580</i> which encodes a putative homoserine dehydrogenase (Hom2), and upstream of <i>CD1582</i> encoding a putative histodinidol dehydrogenase (HisD). Transcription of <i>cdeM</i> is predicted (by RNA-Seq) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref028" target="_blank">28</a>] to be under the control of a σ^K-regulated promoter immediately upstream of <i>cdeC</i>; however, transcription of <i>hom2</i> and <i>hisD</i> is not dependent on sporulation [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref028" target="_blank">28</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref030" target="_blank">30</a>]. The main repeats in CdeC and CdeM are shown in the magnification of the predicted protein primary sequence in color and described in the text. Blue spirals and arrows indicate the predicted beta sheets and alpha helixes [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref031" target="_blank">31</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.ref032" target="_blank">32</a>]. <b>(B)</b>, Gene neighborhoods of predicted-coding regions whose products have homology to <i>C</i>. <i>difficile</i> 630<i>erm</i> CdeC and CdeM by a blastp search. The diagram is abridged to show only the first neighborhood in each genome for the <i>cdeC</i> locus and <i>cdeM</i> locus. Predicted proteins with homology to <i>C</i>. <i>difficile</i> CdeC and CdeM were clustered by sequence identity (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#ppat.1007199.s003" target="_blank">S3</a> Figs). The percentage of identity with <i>C</i>. <i>difficile</i> 630<i>erm</i> CdeC and CdeM of homologues in other species is shown in green.</p

Transmission electron micrographs of <i>C</i>. <i>difficile cdeM</i> and <i>cdeC</i> spores.

<p><b>(A)</b> Thin sections of <i>C</i>. <i>difficile</i> wild-type, <i>cdeC</i> and <i>cdeM</i> spores were analyzed by transmission electron microscopy as described in the Method section. Representative micrographs of several <i>C</i>. <i>difficile</i> wild-type, <i>cdeC</i> and <i>cdeM</i> spores are shown in the upper panel. Selected individual spores of wild-type with thick and thin exosporium layer are shown. The middle panel shows representative individual spores of the mutant strains <i>cdeC</i> and <i>cdeM</i>. The lower panel shows a magnified view of the thin section of wild-type, <i>cdeC</i> and <i>cdeM</i> spores with thick and thin exosporium layers. Ex, exosporium; Ic, inner coat; Ec, external coat; Cx, cortex. <b>(B)</b> The thickness of the exosporium and outer and inner coat layers of <i>C</i>. <i>difficile</i> wild-type (white bars), <i>cdeC</i> (gray bars) and <i>cdeM</i> (black bars) strains were analyzed by transmission electron microscopy of at least 10 individual spores with an apparent thick-exosporium morphotype. Error bars denote standard errors of the means. Asterisks (*) denote statistical difference at <i>P</i> < 0.05 and (**) denote statistical difference at <i>P</i> < 0.001 respect to wild-type. Scale bars are shown in each figure: the bars in the upper panels represent 1 nm, middle panel 100 nm and the bars in the lower panels represent 200 nm. <b>(C)</b> The surface accessibility of CdeC on <i>C</i>. <i>difficile</i> 630<i>erm</i> wild-type and <i>cdeC</i> mutant spores was analyzed by immunofluorescence with rat anti-CdeC serum as described in Methods section. <b>(D)</b> The surface accessibility of CdeM on <i>C</i>. <i>difficile</i> 630<i>erm</i> wild-type and <i>cdeM</i> mutant spores was analyzed by immunofluorescence with rabbit anti-CdeM spores as described in Methods section.</p

Relative abundance of Flag-fusions of spore coat and exosporium proteins in <i>C</i>. <i>difficile</i> spores in the absence of CdeC and CdeM^a.

<p>Relative abundance of Flag-fusions of spore coat and exosporium proteins in <i>C</i>. <i>difficile</i> spores in the absence of CdeC and CdeM<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007199#t001fn001" target="_blank">^a</a>.</p