Uropathogenic Escherichia coli Modulates Immune Responses and Its Curli Fimbriae Interact with the Antimicrobial Peptide LL-37

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms

Antimicrobial peptides and proteins in host-microbe interaction and immediate defense

Antimicrobial peptides and proteins (AMPs) are effector molecules of innate immunity and are capable to kill a broad spectrum of microbes, i.e. bacteria, fungi and viruses. They are widespread in nature and have been found in almost all species of the animal kingdom, as well as in plants. The mammalian repertoire of antimicrobial peptides includes the defensins and the cathelicidins. Furthermore, several of the antimicrobial proteins are members of the S100 family. AMPs are predominantly expressed at exposed surfaces and in specific white blood cells. In the first part of this thesis, the presence of AMPs in material derived from the skin and gut of the fetus/neonate was investigated. Interestingly, the levels of AMPs of the gut were found to be induced after birth. Apart from being antimicrobial, AMPs are important in immunity by exhibiting additional functions, such as chemotactic, mitogenic and wound healing activities. Hence, in the second part of this thesis, novel immunological functions of several AMPs were discovered, further supporting their multifunctional role in host defense. Vernix caseosa (vernix) is a lipid-rich material covering the skin of newborn babies and is suggested to serve as an anti-infective substance, both for the fetus during the last trimester of gestation, and for the neonate during the first days of life. In paper I, protein extracts of vernix were found to exhibit antimicrobial activity against three bacterial strains and one fungal strain. In a proteomic approach, we identified approximately 20 proteins possessing diverse effects implicated in innate immunity, such as opsonizing, anti-parasitic and anti-protease activities. In paper II, we reported that protein extracts of meconium and neonatal feces exhibit antimicrobial activity against gram-positive and gram-negative bacteria. We also identified several AMPs in both these extracts that most likely contribute to the observed antimicrobial activities. Interestingly, the levels of some AMPs were found at higher levels in feces than in meconium, suggesting an induction or release of AMPs after birth upon colonization and breast feeding. As demonstrated in paper III, physiological concentrations of hydrophilic components of breast milk induce the expression of the cathelicidin LL-37 in colonic epithelial cells after 48 h of stimulation. This novel role of breast milk may contribute to the protection against infections in the gut of breast-fed babies. In paper IV of this thesis, LL-37 was found to inhibit biofilm formation of uropathogenic Escherichia coli. This inhibition is likely mediated by binding of LL-37 to the major curli subunit CsgA, preventing polymerization of CsgA to curli fibers. We demonstrated that biofilm of E. coli increased the resistance to cathelicidins, indicating that the inhibition of biofilm is a crucial anti-infective mechanism, making the bacteria more susceptible to killing. Furthermore, in paper V, we reported that neutrophil defensins 1-3 (HNP1-3) and heparin binding protein (HBP) enhanced macrophage phagocytosis of IgG-opsonized bacteria. The mechanism mediating this effect was found to be the release of IFNgamma and TNFalpha, activating the macrophages in an autocrine manner, ultimately resulting in upregulation of Fc-receptors. The anti-biofilm effect and the enhanced phagocytosis mediated by AMPs are novel effects, demonstrating that AMPs contribute to additional defense strategies that most likely are important in combating infections

Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant

Somatostatin and its 2A receptor in dorsal root ganglia and dorsal horn of mouse and human : expression, trafficking and possible role in pain

Background: Somatostatin (SST) and some of its receptor subtypes have been implicated in pain signaling at the spinal level. In this study we have investigated the role of SST and its sst2A receptor (sst2A) in dorsal root ganglia (DRGs) and spinal cord.Results: SST and sst2A protein and sst2 transcript were found in both mouse and human DRGs, sst2A-immunoreactive (IR) cell bodies and processes in lamina II in mouse and human spinal dorsal horn, and sst2A-IR nerve terminals in mouse skin. The receptor protein was associated with the cell membrane. Following peripheral nerve injury sst2A-like immunoreactivity (LI) was decreased, and SST-LI increased in DRGs. sst2A-LI accumulated on the proximal and, more strongly, on the distal side of a sciatic nerve ligation. Fluorescence-labeled SST administered to a hind paw was internalized and retrogradely transported, indicating that a SST-sst2A complex may represent a retrograde signal. Internalization of sst2A was seen in DRG neurons after systemic treatment with the sst2 agonist octreotide (Oct), and in dorsal horn and DRG neurons after intrathecal administration. Some DRG neurons co-expressed sst2A and the neuropeptide Y Y1 receptor on the cell membrane, and systemic Oct caused co-internalization, hypothetically a sign of receptor heterodimerization. Oct treatment attenuated the reduction of pain threshold in a neuropathic pain model, in parallel suppressing the activation of p38 MAPK in the DRGs. Conclusions: The findings highlight a significant and complex role of the SST system in pain signaling. The fact that the sst2A system is found also in human DRGs and spinal cord, suggests that sst2A may represent a potential pharmacologic target for treatment of neuropathic pain.16 page(s

Induction of CAMP gene transcript in different cell lines stimulated with lactose.

<p>(<b>A</b>) HT-29 cells were stimulated with 10–70 g/l lactose for 48 h and <i>CAMP</i> gene transcript was monitored. At 20 g/l a 3.6-fold expression was observed that increased dose-dependently up to 11.3-fold at 70 g/l. (<b>B</b>) HT-29 cells were stimulated with 60 g/l lactose for 4, 24 and 48 h. After 24 h and 48 h an 8- and 17-fold induction of <i>CAMP</i> gene transcript was observed. No induction of <i>CAMP</i> gene was observed at 4 h. (<b>C</b>) T84 cells were stimulated with 60 g/l lactose for 4, 24 and 48 h. A 3.5-fold enhanced level of <i>CAMP</i> gene transcript was observed after 24 h and was 11.3-fold after 48 h. (<b>D</b>) THP-1 monocytes (black line) and differentiated macrophage-like THP-1 cells (grey line) were stimulated with 60 g/l lactose for 4, 24 and 48 h. In monocytes, 7.2 and 25.7-fold induction of <i>CAMP</i> gene transcript was detected after 24 h and 48 h, respectively. The macrophage-like cells exhibited a 13.5-fold induction of <i>CAMP</i> gene transcript after 24 h that declined to 5.6-fold after 48 h. (<b>A–D</b>) Displays the mean and SD of five independent experiments in duplicate.</p

Relative inductions of CAMP gene transcript by colostrum, transitional milk, mature milk and infant formulas.

<p>(<b>A</b>) HT-29 cells were stimulated for 48 h with 50 g/l hydrophilic fractions of breast milk from different lactation periods. The median of the relative induction of <i>CAMP</i> gene transcript was 2.5-fold for colostrum (1–3 days postpartum, pp), 2.1-fold for transitional milk (4–10 days pp) and 3.9-fold for mature milk (11- days pp) samples. The median of 50 g/l hydrophilic fraction of infant formulas was 2.2, but with a high variability in <i>CAMP</i> gene induction. (<b>B</b>) HT-29 cells were stimulated with the hydrophilic fraction of breast milk collected from one mother from day 7 to 19 pp. A positive linear correlation was observed between <i>CAMP</i> gene transcript induction and time pp (R² = 0.5728, p-value 0.043). (<b>A and B</b>) Each sample is performed in triplicates.</p

Isolation of the CAMP gene inducing component.

<p>(<b>A</b>) Heat treatment (100°C) of the hydrophilic fraction of breast milk for 30 min did not affect <i>CAMP</i> gene inducing capacity in HT-29 cells after 48 h of stimulation. Breast milk components were separated into high and low molecular weight components (more or less than 10 kDa) and the <i>CAMP</i> gene inducing capacity of breast milk was retained only in the low molecular weight fraction (≤10 kDa). Displays the mean and SD of at least three independent experiments in triplicate. (<b>B</b>) The low molecular fraction was subjected to cationic exchange and the chromatographic fractions were used for stimulation of HT-29 cells for 48 h. Material in fraction 29, eluted at 4% buffer B, resulted in a 10-fold induction of <i>CAMP</i> gene transcript. (<b>C</b>) Material in fraction 29, from (<b>B</b>), was separated by size exclusion chromatography and obtained fractions were assayed for <i>CAMP</i> gene transcript in HT-29 cells after 48 h of stimulation. A 6.5-fold induction of <i>CAMP</i> gene was observed by stimulation with material from fraction 42. (<b>B–C</b>) The X-axes denote the elution volume. The grey bars representing the activity are shown as a mean of the fold induction performed in triplicate.</p

Characterization of the CAMP gene inducing component.

<p>(<b>A</b>) The intact molecular mass of the most abundant components in fraction 42 was 365.1 and 707.2 corresponding to a sodiated monomer of a disaccharide [342.1+ Na]⁺ and a sodiated dimer of a disaccharide [2×342.1+ Na]⁺, respectively. The mass of 343.1 corresponded to a protonated disaccharide [342.1+H]⁺. (<b>B</b>) Fragmentation of the 707.2 component resulted in a peak of 365.1, supporting the presence of disaccharides <i>i.e.</i> [342+Na]⁺. (<b>C</b>) Fragmentation of the 343.1 peak resulted in two peaks of 325.1 and 163.1, corresponding to a disaccharide and a monosaccharide with the elimination of water, respectively. (<b>D</b>) Top: ¹H-NMR spectrum in D₂O (at 20°C) of the pooled fractions 40–42. Bottom: ¹H-NMR spectrum in D₂O (at 20°C), of the equilibrated α/β mixture of lactose. These results establish lactose as the major component in the active fractions.</p

The hydrophilic fraction of breast milk induces the CAMP gene in HT-29 cells.

<p>(<b>A</b>) HT-29 cells were stimulated with 5 or 50 g/l hydrophilic fraction of breast milk for 4, 24 or 48 h. After 48 h a 10.8-fold increase of <i>CAMP</i> gene transcript was observed with 50 g/l. (<b>B</b>) Stimulation of HT-29 cells with the hydrophobic fraction of breast milk did not significantly induce <i>CAMP</i> gene expression. (<b>A–B</b>) Show means and standard deviations (SD) of at least three independent experiments performed in triplicates. (<b>C</b>) Western blot analysis showed induction of <i>CAMP</i> gene expression at the protein level. A low constitutive expression of the LL-37 proform hCAP-18 (17 kDa) was observed in the supernatants of unstimulated cells. hCAP-18 concentration increased after stimulation with both 5 and 50 g/l of the hydrophilic fraction with the highest increase after 48 h with 50 g/l. Control (ctrl) was 50 g/l of hydrophilic fraction mixed with cell medium from 48 hours unstimulated cells. Band intensities were calculated from one representative Western blot analysis using Image J and normalized to the band intensity of supernatants from cells propagated for 4 h in medium (US 4).</p

Galactose and lactose levels in feces collected from neonates 1 or 4 weeks after birth. Displays the mean of samples analyzed in triplicate<b>.</b>

<p>Galactose and lactose levels in feces collected from neonates 1 or 4 weeks after birth. Displays the mean of samples analyzed in triplicate<b>.</b></p