Bordetella pertussis Naturally Occurring Isolates with Altered Lipooligosaccharide Structure Fail To Fully Mature Human Dendritic Cells

Abstract

bBordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.T he innate immune system is the first line of defense against invading pathogens. In order to recognize these microorganisms, innate immune cells express multiple pathogen recognition receptors (PRR) consisting of various receptor families, including the most-studied Toll-like receptors (TLRs) (1). Evasion of this first recognition can be critical for the pathogen establishing infection. Additionally, the innate immune system is essential for the induction and regulation of the adaptive response. For example, dendritic cells (DCs) can produce interleukin-12p70 (IL12p70), which is associated with the differentiation of T helper 1 (Th1) cells or cytotoxic T cells (2, 3) and IL-1␤, IL-6, and IL-23, which are required for differentiation and survival of Th17 cells (4, 5). Pathogen adaptation, through changes in the structure or expression of molecules which interact with the host, might allow the pathogen to escape the host immune responses.Pertussis, also referred to as whooping cough, is a humanspecific respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. It has been shown that B. pertussis is able to activate both TLR2 and TLR4 signaling on innate immune cells, which is essential for inducing protective immunity against this bacterium (6-9). A well-defined ligand for TLR4 is lipopolysaccharide (LPS), which is produced by Gram-nega...

Emerging Bordetella pertussis Strains Induce Enhanced Signaling of Human Pattern Recognition Receptors TLR2, NOD2 and Secretion of IL-10 by Dendritic Cells

“…pertussis strains has previously been reported to alter TLR4 signalling and DC activation [25, 40]. We observed no differences in TLR4 activation by strains from the three periods indicating no structural modifications of the LOS molecule from these B .…”

“…PRR signaling leads to the expression of SEAP, which can be detected in culture supernatants upon addition of the substrate Quanti-Blue (InvivoGen). The HEK-Blue cells were cultured as previously described [25]. In short, HEK-Blue cells were stimulated in a volume of 200 μl in 96-wells plates for 24 hours according to the manufacturer’s instructions.…”

“…MoDcs were purified from peripheral blood derived from healthy donors and cultured as described previously [25]. Briefly, after peripheral blood mononuclear cell isolation using a Lymphoprep (Nycomed, Zurich, Switzerland) gradient, monocytes were collected by using MACS in combination with anti-CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany).…”

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination

“…The expression of Lbp, Cd14, and Tlr2 indicate the involvement of Toll-like receptors 2 and 4 (TLR2 and TLR4), similar to what was observed with whole-cell PV and live bacteria. [29][30][31] Interestingly, Tlr7 and Nlrp3 were expressed, indicating activation of intracellular PRRs by omvPV. Subsequently, genes downstream of PRRs, including MyD88 and nuclear factor-kB, were expressed.…”

Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor

“…Monocytes were purified from peripheral blood derived from healthy donors as described previously 22 . Briefly, peripheral blood mononuclear cells were isolated by Lymphoprep (Nycomed, Zurich, Switzerland) gradient centrifugation at 1000 × g for 30 min.…”