Environmental Burkholderia cenocepacia Strain Enhances Fitness by Serial Passages during Long-Term Chronic Airways Infection in Mice

Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656(T). Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts

Diagnóstico de la producción de plantas forestales en los viveros de la Municipalidad de Guatemala; evaluación del enraizamiento de matilisguate (Tabebuia rosea bertol) y servicios realizados en el Vivero Municipal Acatán, Zona 16, Ciudad De Guatemala, Guatemala, C.A.

El presente trabajo de investigación se orientó hacia la producción de plantas forestales de los viveros de la Municipalidad de Guatemala, referente a distintos criterios de selección del material al momento de plantarse en campo, así como principales modos de propagación que se utilizan en los viveros para abastecer a los distintos proyectos de jardinización y arborización urbana. Mediante el diagnóstico se puedo conoce la situación actual de los sistemas de producción de plantas forestales en los viveros, se determinaron las principales características de las especies forestales que se utilizan para arborización urbana, jardinización, criterios de selección y modos de propagación, encontrándose como principal medio de propagación la semilla y no otras alternativas que agilicen la obtención masal de plántulas de mayor tamaño en el menor tiempo posible. La investigación se dirigió a establecer el un método alternativo para la propagación vegetativa de una especie ampliamente utilizada para arborización urbana: Tabebuia rosea bertol. Se evaluó la respuesta al enraizamiento en vástago, en función de tres concentraciones del ácido Indol3Butírico y tres diámetros de estaca, en plantas en fase de vivero con material juvenil para propagar. La investigación tuvo una duración de 4 meses. El estudio se realizó en el Huerto y Vivero Urbano Municipal Acatán zona 16, ciudad de Guatemala. El resultado de la investigación, demostró que existe respuesta a la propagación vegetativa por la especie bajo estudio, la respuesta fue mayor para el caso de propagación por vástago de 2.5cm. de diámetro y 2500ppm del regulador. Los resultados se midieron en presencia de callo, de raíces, y de brotes; además, peso seco de raíces, altura de la planta. Este resultado es de importancia ya que permite obtener plantas de matilisguate de mayor tamaño en menos tiempo y esto se vería reflejado en el arbolado urbano

An intestinal Th17 subset is associated with inflammation in crohn's disease and activated by adherent-invasive Escherichia coli (aiec)

IFNγ-producing ex-Th17-cells ("Th1/17") were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterized a novel, potentially colitogenic subset of Th17-cells in the intestine of patients with Crohn's Disease (CD). Human Th17-cells expressing CCR5 ("pTh17") co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17-cells. pTh17-cells, but not Th1/17-cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5(-)Th17-cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17-cells in the intestine. Importantly, intestinal pTh17-cells were selectively activated by adherent-invasive Escherichia coli (AIEC), but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from DC. Intestinal CCR5 +Th1/17-cells responded instead to Cytomegalovirus and were reduced in UC, suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17-cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role

Positive Signature-Tagged Mutagenesis in Pseudomonas aeruginosa: Tracking Patho-Adaptive Mutations Promoting Airways Chronic Infection

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection

Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes

Hematopoietic stem and progenitor cells (HSPCs) are multipotent stem cells that have been harnessed as a curative therapy for patients with hematological malignancies. Notably, the discovery that HSPCs are endowed with immunoregulatory properties suggests that HSPC-based therapeutic approaches may be used to treat autoimmune diseases. Indeed, infusion with HSPCs has shown promising results in the treatment of type 1 diabetes (T1D) and remains the only “experimental therapy” that has achieved a satisfactory rate of remission (nearly 60%) in T1D. Patients with newly diagnosed T1D have been successfully reverted to normoglycemia by administration of autologous HSPCs in association with a non-myeloablative immunosuppressive regimen. However, this approach is hampered by a high incidence of adverse effects linked to immunosuppression. Herein, we report that while the use of autologous HSPCs is capable of improving C-peptide production in patients with T1D, ex vivo modulation of HSPCs with prostaglandins (PGs) increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. Surprisingly, CXCR4 was upregulated as well, which could enhance HSPC trafficking toward the inflamed pancreatic zone. When tested in murine and human in vitro autoimmune assays, PG-modulated HSPCs were shown to abrogate the autoreactive T cell response. The use of PG-modulated HSPCs may thus provide an attractive and novel treatment of autoimmune diabetes

Image_1_Phagocytosis and Epithelial Cell Invasion by Crohn’s Disease-Associated Adherent-Invasive Escherichia coli Are Inhibited by the Anti-inflammatory Drug 6-Mercaptopurine.pdf

<p>Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn’s disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli, might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.</p

Impact of chronic pulmonary infection with Pseudomonas aeruginosa on transfection mediated by viral and nonviral vectors

Pseudomonas aeruginosa plays a crucial role in the lung pathology of cystic fibrosis (CF). We showed that acute infection with P. aeruginosa has a substantial impact on gene transfer into lung epithelial cells mediated by polyplexes. As an extension of those studies we report here on the effect of chronic pulmonary infection with P. aeruginosa on transfection of lung epithelial cells by viral and nonviral vectors. As an in vivo model of the persistent chronic infection in patients with CF we used C57BL/6 mice intratracheally infected with P. aeruginosa encapsulated in agar beads. Two weeks after infection the presence of viable bacteria in the lungs was confirmed, mostly in the bronchial lumen. In lung tissue sections stained with hematoxylin and eosin, extensive inflammatory infiltrations were found. At that time point the mice received an intratracheal dose of luciferase gene complexed with either Lipofectamine (Lf), a GL67 lipid mixture (GL67), or polyethylenimine (PEI) or with lentivirus (LV) as a carrier system. Luciferase activity was determined by a luminescence assay in supernatants of lung homogenates. The transfection level induced by PEI/DNA polyplexes complexed with serum albumin was decreased in infected mice. Lf-mediated transfection was almost completely blocked in infected mice. Transfection levels in mice treated with LV or plain PEI/DNA polyplexes were unchanged in infected animals as compared with control mice. The only carrier that displayed a clearly increased transfection level in infected mice was the GL67 lipid mixture, which is tentatively ascribed to the presence of polyethylene glycol in this carrier

Inactivation of the Pyrimidine Biosynthesis <i>pyrD</i> Gene Negatively Affects Biofilm Formation and Virulence Determinants in the Crohn’s Disease-Associated Adherent Invasive <i>Escherichia coli</i> LF82 Strain

In Crohn’s disease (CD) patients, the adherent-invasive Escherichia coli (AIEC) pathovar contributes to the chronic inflammation typical of the disease via its ability to invade gut epithelial cells and to survive in macrophages. We show that, in the AIEC strain LF82, inactivation of the pyrD gene, encoding dihydroorotate dehydrogenase (DHOD), an enzyme of the de novo pyrimidine biosynthetic pathway, completely abolished its ability of to grow in a macrophage environment-mimicking culture medium. In addition, pyrD inactivation reduced flagellar motility and strongly affected biofilm formation by downregulating transcription of both type 1 fimbriae and curli subunit genes. Thus, the pyrD gene appears to be essential for several cellular processes involved in AIEC virulence. Interestingly, vidofludimus (VF), a DHOD inhibitor, has been proposed as an effective drug in CD treatment. Despite displaying a potentially similar binding mode for both human and E. coli DHOD in computational molecular docking experiments, VF showed no activity on either growth or virulence-related processes in LF82. Altogether, our results suggest that the crucial role played by the pyrD gene in AIEC virulence, and the presence of structural differences between E. coli and human DHOD allowing for the design of specific inhibitors, make E. coli DHOD a promising target for therapeutical strategies aiming at counteracting chronic inflammation in CD by acting selectively on its bacterial triggers