Persistence and survival of NTHi in the lungs
After adhering successfully to AECs, NTHi must still obtain
nutrients and continue evading host defenses.,NTHi have absolute growth requirements for iron, but are unable
to synthesize heme, which is essential for their survival. Consequently,NTHi has multiple core and accessory genes producing proteins
expressed on the cell surface to acquire iron from free heme and
hemoglobin sequestrators, such as hemopexin and
haptoglobin.77 However, the exact mechanisms remain
poorly understood because of the number, diversity and functional
redundancy of the encoding genes. Recent studies identified that some of
these genes are phase-variably expressed and rapidly acquire point
mutations during persistent infection, resulting in accumulated amino
acid changes in surface exposed regions of iron acquisition proteins,
suggesting these are survival strategies following immune selection
pressures.79 Other OMPs, such as P2, also undergo
antigenic drift with key amino acid sequence changes occurring in key
epitopes, which enable NTHi to persist despite the presence of
potentially protective antibodies.
NTHi utilize phase variation to facilitate adaptation and
survival within multiple niches throughout the respiratory tract. Phase
variation is the random and reversible switching of gene expression,
which permits reversible loss or gain of surface structures, such as
adhesins (HMW1 and HMW2), LOS and iron-acquisition proteins (heme
receptors and hemagglutinating pili).81 Phase-variable
genes have changes in the length of simple sequence repeats and when
located in the open reading frame of a gene this variation in length
determines if the encoded protein is either expressed (ON) or not (OFF).
Phase-variable DNA methyltransferases also control the expression of
multiple genes involved in colonization and survival within different
ecological niches and such systems are termed phasevarions to describe
phase-variable regulons.81 Taken together, these
random changes in gene expression enable NTHi to generate
phenotypic variants, which are better suited to the local lung
environment or more able to evade host defenses.
Widely regarded as an extra-cellular pathogen, NTHi can access
and survive in the paracellular spaces between AECs due to the adherence
and invasion protein, HMW1,85 and following
downregulation of cadherin and claudin proteins responsible for
epithelial cell-to-cell interactions and epithelial tight junction
integrity, respectively.86 As a further survival
strategy, NTHi can also invade mononuclear cells and AECs by
utilizing multiple virulence factors, including IgaA protease, LOS via
the platelet-activating factor receptor leading to cytoskeletal
remodeling, several adhesins, including protein D, Hap, or P1 fimbrae
binding to the CEACAM-1 receptor and the phase-variable adhesin HMW1 and
protein E binding to vitronectin.69,85,87 Under iron
restricted conditions within the lungs, NTHi enter AECs by
macropinocytosis to form bacterial communities within the
cytoplasm.88 Once inside the cell, NTHi must
adapt to the low pH and increased oxidative state within the phagosome
of macrophages or the limited nutrient availability within the AEC
cytoplasm. Relatively little is known of how NTHi survives and
traffics within this environment. Transcriptional factors, such as the
ferric uptake regulator cassette which regulates the expression of
multiple genes controlling the uptake and utilization of iron, and IgaB
that cleaves lysosomal-associated membrane protein-1, appear to be
important, and NTHi can survive for at least 72-hours inside
AECs.
Furthermore, lung explant cultures from COPD and CF patients undergoing
transplantation for end-stage lung disease revealed that despite
negative sputum and lung tissue cultures, NTHi was detected
widely throughout the lung parenchyma and lower airway tissue sections
by immunoperoxidase staining and polymerase chain reaction assays. While
most NTHi were detected in clusters in extracellular and paracellular
locations, in CF patients they were found within macrophages too. The
discrepancy between culture and non-culture results was attributed to
pre-operative antibiotics and the inhibitory effects ofPseudomonas aeruginosa upon NTHigrowth.91 However, paracellular and intracellular
locations of NTHi and biofilms may also be important.