Targeted Delivery of Narrow-Spectrum Protein Antibiotics to the Lower Gastrointestinal Tract in a Murine Model of Escherichia coli Colonization.

Bacteriocins are narrow-spectrum protein antibiotics that could potentially be used to engineer the human gut microbiota. However, technologies for targeted delivery of proteins to the lower gastrointestinal (GI) tract in preclinical animal models are currently lacking. In this work, we have developed methods for the microencapsulation of Escherichia coli targeting bacteriocins, colicin E9 and Ia, in a pH responsive formulation to allow their targeted delivery and controlled release in an in vivo murine model of E. coli colonization. Membrane emulsification was used to produce a water-in-oil emulsion with the water-soluble polymer subsequently cross-linked to produce hydrogel microcapsules. The microcapsule fabrication process allowed control of the size of the drug delivery system and a near 100% yield of the encapsulated therapeutic cargo. pH-triggered release of the encapsulated colicins was achieved using a widely available pH-responsive anionic copolymer in combination with alginate biopolymers. In vivo experiments using a murine E. coli intestinal colonization model demonstrated that oral delivery of the encapsulated colicins resulted in a significant decrease in intestinal colonization and reduction in E. coli shedding in the feces of the animals. Employing controlled release drug delivery systems such as that described here is essential to enable delivery of new protein therapeutics or other biological interventions for testing within small animal models of infection. Such approaches may have considerable value for the future development of strategies to engineer the human gut microbiota, which is central to health and disease.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7.

Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health problem, as they are problematic to treat. Stx production is a key virulence factor associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result in the development of haemolytic uremic syndrome in infected patients. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants.

Convergent evolution of pathogenicity islands in helper cos phage interference.

Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'.

Immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota activate retinal microglia in mouse models.

Although its actual role in the progression of degenerative processes is not fully known, the persistent activated state of retinal microglia and the concurrent secretion of inflammatory mediators may contribute to neuronal death and permanent vision loss. Our objective was to determine whether non-ocular conditions (immunosuppression and peripheral inflammation) could lead to activation of retinal microglia. Mouse models of immunosuppression induced by cyclophosphamide and/or peripheral inflammation by chemically induced sublethal colitis in C57BL/6J mice were used. Retinal microglia morphology, spatial distribution and complexity, as well as MHCII and CD11b expression levels were determined by flow cytometry and confocal immunofluorescence analysis with anti-CD11b, anti-IBA1 and anti-MHCIIRT1B antibodies. Retinas of mice with double treatment showed changes in microglial morphology, spatial distribution and expression levels of CD11b and MHCII. These effects were higher than those observed with any treatment separately. In addition, we also observed in these mice: (i) translocation of endogenous bacteria from gut to liver, and (ii) upregulation of TLR2 expression in retinal microglia. Using a mouse model of immunosuppression and gut colonization by Candida albicans, translocation of fungal cells was confirmed to occur in wild type and, to a higher extent, in TLR2 KO mice, which are more susceptible to fungal invasion; interestingly microglial changes were also higher in TLR2 KO mice. Hence, non-ocular injuries (immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota) can activate retinal microglia and therefore could affect the progression of neurodegenerative disorders and should be taken into account to improve therapeutic options.

TLR2 modulates gut colonization and dissemination of Candida albicans in a murine model.

Invasive candidiasis often arises from translocation of endogenous yeasts from the gastrointestinal tract to the bloodstream. Here we describe that both wild type and TLR2-/- mice strains, orally administered with Candida albicans yeasts, display similar sustained high level of gut colonization when oral antibacterial treatment is present, while removal of antibiotic treatment causes a progressive clearance of yeasts in control but not in TLR2-/- mice. Fungal invasion of internal organs, following immunosuppression of colonized mice, was increased in TLR2-/- mice. These results point out to a role of TLR2 in gut protection against colonization and endogenous invasion by C. albicans.

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

Bacteriophage-mediated spread of bacterial virulence genes.

Bacteriophages are types of viruses that infect bacteria. They are the most abundant and diverse entities in the biosphere, and influence the evolution of most bacterial species by promoting gene transfer, sometimes in unexpected ways. Although pac-type phages can randomly package and transfer bacterial DNA by a process called generalized transduction, some mobile genetic elements have developed elegant and sophisticated strategies to hijack the phage DNA-packaging machinery for their own transfer. Moreover, phage-like particles (gene transfer agents) have also evolved, that can package random pieces of the producing cell's genome. The purpose of this review is to give an overview of some of the various ways by which phages and phage-like particles can transfer bacterial genes, driving bacterial evolution and promoting the emergence of novel pathogens.

Retinal microglia are activated by systemic fungal infection.

PURPOSE: We determined whether systemic fungal infection could cause activation of retinal microglia and, therefore, could be potentially harmful for patients with retinal degenerative diseases. METHODS: Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by confocal immunofluorescence and flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII, and anti-CD45 antibodies. RESULTS: Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial relocation in retinal layers. CONCLUSIONS: As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration, or retinitis pigmentosa.

ß-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and p38.

ß-Adrenoceptors (ß-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of ß-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that ß-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline ß-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after ß-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium ß-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.

Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases.

Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. In this report, we have characterized one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages--i.e., it has both pac and cos sites--a configuration that has not hitherto been described for any mobile element, phages included--and uses the two different phage-coded TerSs. To our knowledge, this is the first example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.

Relación entre modelado miocárdico y función diastólica en pacientes con hipertensión esencial / Relationship between myocardial modelling and diastolic function in patients with essential hypertension

Fundamento y objetivo: Analizar la relación entre las citoquinas antiapoptóticas, sFas y receptor soluble tipo 1 del factor de necrosis tumoral (sTNF-R1) con el fragmento aminoterminal del propéptido de procolágeno tipo iii (PIIINP) y tipo i (PINP) y la función diastólica en la hipertensión arterial (HTA). Pacientes y método: Se estudió un grupo de 253 pacientes con HTA, con una edad media (DE) de 60 (13) años; 139 eran varones. Se les realizó un examen físico, estudio eco-Doppler y determinación sérica de las moléculas. Resultados: Los valores séricos de PINP y PIIINP estuvieron elevados en pacientes hipertróficos frente a no-hipertróficos (media de 41 [extremos 31-52] frente a 35 [28-47] μg/l, p=0,010; y 4,33 [3,71-5,29] frente a 3,98 [3,49-4,58] μg/l, p=0,005, respectivamente). Además, se encontraron elevadas las concentraciones de sFas y sTNF-R1 (media de 1,47 [extremos 1,2-1,77] frente a 1,37 [1,1-1,59] ng/ml, p=0,012; y 466 [331-657] frente a 317 [260-427] pg/ml, p<0,0001, respectivamente). Por otra parte, los valores de PIIINP se relacionaron con sFas (r=0,386, p<0,0001) y sTNF-R1 (r=0,298, p<0,001); también se asoció PINP con estas citoquinas (r=0,158, p=0,011 y r=0,241, p<0,0001, respectivamente). En el análisis multivariable, sFas (p<0,0001) y sTNF-R1 (p<0,0001) fueron factores independientes de PIIINP. Por último, las concentraciones de los marcadores se relacionaron significativamente con los parámetros de función diastólica del ventrículo izquierdo. Conclusión:Las concentraciones de procolágeno y de citoquinas antiapoptóticas estuvieron elevadas en pacientes con hipertrofia ventricular. Además, sFas y sTNF-R1 son factores independientes de los valores en suero de PIIINP y se relacionan con los parámetros de función diastólica ventricular (AU)

Background and objectives: To analyze the relationship between sFas and soluble TNF receptor 1 (sTNF-R1) with type iii (PIIINP) and i (PINP) amino-terminal propeptide procollagens, and diastole in hypertension (HT). Patients and methods: A group of 253 Caucasian asymptomatic hypertensive patients (age 60±13 years, 139 males) were studied, in whom a physical examination, laboratory analyses (determination of serum PIIINP, PINP, sFas and by radioimmunoassay and ELISA, respectively), and echo-Doppler study were performed. Results: Serum PINP and PIIINP were increased in left ventricular hypertrophy compared to non-hypertrophy [41 (31-52) vs. 35 (28-47) μg/l, P=.010; and 4.33 (3.71-5.29) vs. 3.98 (3.49-4.58) μg/l, P=.005, respectively]. Furthermore, sFas and sTNF-R1 were also elevated [1.47 (1.2-1.77) vs. 1.37 (1.1-1.59), P=.012; and 466 (331-657) vs. 317 (260-427) μg/l, P<.0001, respectively]. Moreover, serum PIIINP was associated with sFas (r=.386, P<.0001) and sTNF-R1 (r=.298, P<.001); PINP was also associated with these cytokines (r=0.158, P=.011 and r=.241, P<.0001, respectively). Multivariable analyses included sFas (P<.0001) and sTNF-R1 (P<.0001) as independent factors related with serum PIIINP. Finally, marker concentrations were significantly related with left ventricular diastolic function parameters. Conclusion: Procollagen and anti-apoptotic cytokine levels were increased in our hypertrophic patients. Furthermore, sFas and sTNF-R1 are independent related factors of serum PIIINP. Diastolic parameters were associated with myocardial fibrosis and anti-apoptotic cytokines (AU)

MMP-2 and sTNF-R1 Variability in Patients with Essential Hypertension: 1-Year Follow-Up Study.

The aim of this study is to analyze MMP-2 and sTNF-R1 variability, potent predictors of cardiovascular events, in stable hypertensive patients during a 12-month followup. 234 asymptomatic patients (age 60 ± 13, 136 male) out of 252 patients with essential hypertension were followed up. MMP-2 and sTNF-R1 were measured at baseline and after 12 months (stage I). To compare MMP-2 and sTNF-R1 levels over time interval, we used the statistical method of Bland-Altman. MMP-2 and sTNF-R1 reproducibility was good in our patients for the two intervals with a coefficient of reproducibility of 8.2% and 11.3%, respectively. The percentages of patients within 1.96 × standard deviation of the mean were 93.6% and 92.7%. An elevated coefficient of correlation was obtained for MMP-2, basal versus stage I (r = 0.55, P < 0.0001) and for sTNF-R1 (r = 0.75, P < 0.0001). There is good stability in MMP-2 and sTNF-R1 levels in a followup study of patients with stable hypertension. As a consequence, assessment of its concentrations may be a useful tool for monitoring the follow-up of these patients. Measured variations in MMP-2 and sTNF-R1 levels, exceeding 8.2% and 11.3%, respectively, may indicate an increase in cardiovascular risk, thus, could be used to optimizing treatment than blood pressure control alone.

[Relationship between myocardial modelling and diastolic function in patients with essential hypertension]. / Relación entre modelado miocárdico y función diastólica en pacientes con hipertensión esencial.

BACKGROUND AND OBJECTIVES: To analyze the relationship between sFas and soluble TNF receptor 1 (sTNF-R1) with type iii (PIIINP) and i (PINP) amino-terminal propeptide procollagens, and diastole in hypertension (HT). PATIENTS AND METHODS: A group of 253 Caucasian asymptomatic hypertensive patients (age 60±13 years, 139 males) were studied, in whom a physical examination, laboratory analyses (determination of serum PIIINP, PINP, sFas and by radioimmunoassay and ELISA, respectively), and echo-Doppler study were performed. RESULTS: Serum PINP and PIIINP were increased in left ventricular hypertrophy compared to non-hypertrophy [41 (31-52) vs. 35 (28-47) µg/l, P=.010; and 4.33 (3.71-5.29) vs. 3.98 (3.49-4.58) µg/l, P=.005, respectively]. Furthermore, sFas and sTNF-R1 were also elevated [1.47 (1.2-1.77) vs. 1.37 (1.1-1.59), P=.012; and 466 (331-657) vs. 317 (260-427) µg/l, P<.0001, respectively]. Moreover, serum PIIINP was associated with sFas (r=.386, P<.0001) and sTNF-R1 (r=.298, P<.001); PINP was also associated with these cytokines (r=0.158, P=.011 and r=.241, P<.0001, respectively). Multivariable analyses included sFas (P<.0001) and sTNF-R1 (P<.0001) as independent factors related with serum PIIINP. Finally, marker concentrations were significantly related with left ventricular diastolic function parameters. CONCLUSION: Procollagen and anti-apoptotic cytokine levels were increased in our hypertrophic patients. Furthermore, sFas and sTNF-R1 are independent related factors of serum PIIINP. Diastolic parameters were associated with myocardial fibrosis and anti-apoptotic cytokines.