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.