Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage

Abstract

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.

Figure 1. B. fragilis and B. henselae internalize EPCs. A.

Confocal images of human early EPCs infected with B. henselae, B. fragilis and B. fragilis ΔPSA at 100 MOI. Cells were stained with anti-Bacteroides (red) or with anti-Bartonella (green) specific antibodies after 24 h from infection. B. EPCs coinfected with B. henselae, and B. fragilis or B. fragilis ΔPSA as indicated. A MOI of 100 was used for all bacteria strains. Cells were stained with anti-Bacteroides (red) and with anti-Bartonella (green) specific antibodies after 24 h from infection.

Figure 2. Expression levels of inflammatory genes in infected EPCs. A.

Expression levels of 84 inflammatory genes in EPCs infected with B. henselae, B. fragilis, B. fragilis ΔPSA, B. henselae and B. fragilis, B. henselae and B. fragilis ΔPSA. Differential expression data are evaluated vs uninfected EPCs. B. Bar graph showing the expression of some relevant differentially expressed genes encoding chemokines, cytokines and their receptors in B. fragilis- vs B. fragilis ΔPSA-infected EPCs. Data are shown as relative expression levels (B. fragilis ΔPSA-infected cells = 1). C. and D. Expression levels of representative pro-inflammatory (C) and anti-inflammatory (D) genes highly differentially expressed during coinfection of EPCs, in presence and in absence of PSA. E. and F. Absolute quantification of relevant secreted inflammatory proteins detected by ELISA in the EPCs infected with B. henselae, and coinfected with B. fragilis and B. fragilis ΔPSA.

Figure 3. Murine infection with B. henselae and B. fragilis and detection of bacteria in infected tissues. A.

Scheme of murine infection: three groups of C57BL/6J mice (n = 5) were infected with B. henselae, B. fragilis, B. fragilis ΔPSA respectively and two groups of animals (n = 5) were coinfected with B. henselae and B. fragilis or B. fragilis ΔPSA. B. TEM micrographs of the liver of C57BL/6J mice infected with B. fragilis and B. fragilis ΔPSA respectively, B. henselae and coinfected. C. Immunofluorescence analysis of the livers of animals infected with B. henselae, B. fragilis and coinfected. Livers were sectioned and treated with an anti-B. henselae and a Bacteroides LPS primary antibody followed by TRITC- or FITC-conjugated secondary antibodies respectively. Liver sections from uninfected mice were used as controls (data not shown). Selected merge images of coinfected mice were acquired by confocal microscopy (Magnification: all, X 270). D. Detection of B. henselae gltA gene and B. fragilis frdA gene by nested-PCR from the liver samples of experimentally infected C57BL/6J with B. fragilis and B. fragilis ΔPSA (lane 1–2), B. henselae (lane 3–4), coinfected (lane 5–8), respectively. Genomic DNA extracted from B. henselae and B. fragilis was used as positive control (lane 9).

Figure 4. Morphological analysis of murine coinfected tissue by hematoxylin-eosin staining. A.

Representative microscope images of hematoxylin-eosin staining of liver tissues from each group of mice uninfected, infected with B. henselae, B. fragilis and B. fragilis ΔPSA or coinfected, as detailed. Granulomatous inflammatory infiltrates are predominantly evident in the group of mice infected with B. henselae compared to the group infected with B. henselae and B. fragilis ΔPSA and particularly in the mice coinfected with B. henselae and B. fragilis. Uninfected controls are negative, as well as B. fragilis and B. fragilis ΔPSA groups. B. Representative sections stained with hematoxylin-eosin of aortas from all mice groups as described. In the group of mice infected with B. henselae, minimal lesions are observed in the aorta compared to liver tissues.

Figure 5. Morphological analysis of murine coinfected tissue by immunohistochemistry. A.

Representative images of immunohistochemistry analysis of murine liver samples with an antibody against B. henselae are shown (206X magnification). Bar graphs show the mean of the positive cell number per field (HPF) as indicated. 20 fields per each section were analyzed and bars represent standard deviations. B. Immunohistochemistry analysis of murine aorta samples.

Figure 6. Ultrastructural analysis of murine infected tissue.

Electron microscopy of liver and aorta: A. Portal triad of normal liver. B. Granuloma around a centrilobular vein of the liver infected with B. henselae with a characteristic neutrophil. C. Normal aortic intima and media. D. White cells in the tunica adventitia of aorta infected with B. henselae. E. Centrilobular vein of liver coinfected with B. henselae and B. fragilis. F. Granuloma in liver coinfected with B. henselae and B. fragilis ΔPSA. G. Endothelium bulging of aorta coinfected with B. henselae and B. fragilis. H. Endothelium bulging of aorta coinfected with B. henselae and B. fragilis ΔPSA. I. Centrilobular vein of liver infected with B. fragilis. J. Centrilobular vein of liver infected with B. fragilis ΔPSA. K. Endothelium of aorta infected with B. fragilis. L. Endothelium of aorta infected with B. fragilis ΔPSA.

Figure 7. EPC number of infected mice.

EPC relative number of infected mice versus control mice was measured by FACS analysis. Sca-I/Flk-I double positive cells were considered as murine EPCs. Bars indicate standard errors.