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

Abstract

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.

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates

“…Surprisingly, the mean IC 50 value of the mucosal surface-isolated group was significantly greater than that of the invasive isolate group. Previously, we have shown that low-level serum resistance of oropharyngeal non-typable H. influenzae isolates permits them to establish a lower respiratory tract infection (Nakamura et al, 2011), indicating that low-level serum resistance in some sputum isolates is not surprising. Serum resistance of an encapsulated strain is imparted largely by the presence of the polysaccharide capsule (Moxon & Kroll, 1988), as evidenced by the serum resistance of a bexA 2 and phenotypically unencapsulated strain Eagan with an IC 50 value of 1.20 % (K. L. Nelson and A. L. Smith, unpublished data).…”

A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa

“…To examine whether the disruption of PA4456 affects the membrane stability and integrity, we grew the mutant, the wild type, and the complemented strains on agar media containing various amounts of EDTA (1 to 2.5 mM) with or without 0.5% SDS and compared their sensitivities to polymyxin B (molecular weight ϭ 1,301.6) and vancomycin (molecular weight ϭ 1,485.7). Changed susceptibility to these compounds serves as an indication for disrupted outer membrane integrity (56)(57)(58). EDTA, a chelator of divalent cations, affects the stability and integrity of the outer membrane by destabilizing lipopolysaccharide interactions (49) The results showed no differences between PA4456M and the wild type (data not shown).…”

Outer Membrane Protein P5 Is Required for Resistance of Nontypeable Haemophilus influenzae to Both the Classical and Alternative Complement Pathways

“…Specifically, depletion of complement using cobra venom factor restored virulence to a serum-sensitive NTHi mutant (which could not sialylate its LOS as a result of a deletion of the siaB gene, encoding cytidinemonophospho-N-acetylneuraminic acid synthetase) that was otherwise avirulent in complement-sufficient animals (30). Recently, strains isolated from pulmonary infections were shown to exhibit higher levels of serum resistance than nasopharyngeal isolates (64,65). While the mechanism of complement-mediated defense against NTHi in the lung is not fully understood, defects in complement-mediated phagocytosis of NTHi have been identified with macrophages isolated from patients with COPD in comparison to those of healthy nonsmokers (66), suggesting the importance of opsonophagocytosis in controlling NTHi in pulmonary infections.…”

“…Although the increase in IgM binding to P5 mutants was only 50% over the level of binding to wild-type strains, IgM is very efficient at activating complement compared with IgG, as a single IgM molecule is sufficient to engage the C1 complex and initiate the classical pathway (67). Moreover, surveys of clinical NTHi isolates have revealed a correlation between higher levels of IgM binding and decreased serum resistance (64,65). The epitope targeted by serum IgM on P5 mutants is currently not known; however, IgM that is bactericidal for NTHi in normal human serum is directed primarily against the LOS (68).…”