Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface

Molecular Basis of Increased Serum Resistance among Pulmonary Isolates of Non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies

Characterization of human CD25(+) CD4(+) T cells in thymus, cord and adult blood

CD4(+) CD25(+) regulatory T cells prevent organ-specific autoimmune diseases in various animal models. We analysed human lymphoid tissues to identify similar CD25(+) regulatory T cells. Adult peripheral blood contained two populations of CD4(+) T cells that expressed CD25 at different densities. The larger population (≈ 40%) expressed intermediate levels of CD25 (CD25(+)) and displayed a memory T-cell phenotype (CD45RA(−)/RO(+), CD45RB(low), CD95(+), CD62L(low), CD38(low)). The smaller population of cells (≈ 2%) expressed very high levels of CD25 (CD25(++)). In addition to the activation/memory T-cell antigens mentioned above they also expressed intracellular CD152 (CTLA-4) as well as enhanced levels of cell-surface CD122, similar to the murine CD4(+) CD25(+) regulatory counterpart. To exclude that the CD25(++) cells had not been recently primed by external antigen we analysed cord blood and thymus. CD25(++), CD152(+) and CD122(++) cells were present in paediatric thymus (10% of CD4(+) CD8(−) thymocytes) expressing signs of recent selection (CD69(+)) and in cord blood (5% of CD4(+) cells) where they showed a naive phenotype. In addition, cord blood contained a small population of CD25(+) cells (≈ 2% of CD4 T cells) that were CD152(−) and CD122(low) and displayed signs of activation. Together with published data that CD25(+) CD25(++) cells from the thymus and peripheral blood are regulatory, our results suggest that regulatory CD25(+) T cells leave the thymus in a naïve state and become activated in the periphery