# In vitro invasive ability of X and V factor dependent Haemophilus species across different cell types

Singh, NK 2017 , 'In vitro invasive ability of X and V factor dependent Haemophilus species across different cell types', Research Master thesis, University of Tasmania.

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## Abstract

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic human pathogen responsible for diseases such as exacerbations of chronic bronchitis, community acquired pneumonia, otitis media and occasionally conjunctivitis. H. haemolyticus is closely related to NTHi and shares the same respiratory niche as a commensal, but is not an opportunistic respiratory pathogen. Both NTHi and H. haemolyticus can acquire resistance to β-lactam antibiotics via mutations to the ftsI gene and associated amino acid substitutions in penicillin binding protein 3 (PBP3) and the prevalence of such resistant strains is increasing worldwide. The factors associated with pathogenicity of NTHi are complicated, but can include mucociliary interactions, attachment to respiratory mucosa, evasion of mucosal immunity, and invasion of respiratory epithelial cells. There is significant in vivo and in vitro evidence that NTHi can invade and persist within host epithelial cells leading to the hypothesis that this allows the organism to avoid the normal immune response and establish a persistent reservoir for infection. Despite increased understanding of some mechanisms involved with invasion, the relationship between intracellular NTHi and pathogenesis is still unclear and many studies have shown enormous strain-to-strain variation in the in vitro invasive ability of clinical isolates. One of the limitations in understanding the relationship between intracellular NTHi and pathogenesis is the lack of a standardised model for studying invasion, as a very large range of both respiratory and non-respiratory, and primary and immortal cell lines have been used, often without explanation or justification. It is unclear whether an isolate that shows in vitro invasion in one cell type will be similarly invasive in another cell type, and this makes comparisons between studies very difficult.
The aims of this thesis were to investigate the effect of respiratory cell types and presence of altered PBP3 on invasion rate of NTHi and Haemophilus haemolyticus. To investigate our aims, we established a large collection of NTHi and H. haemolyticus isolates where the identity had been confirmed previously using a validated PCR algorithm as either NTHi (being positive for hpd#3 or fucK and negative for sodC) or H. haemolyticus (positive for sodC and negative for both hpd#3 and fucK. In this working collection, NTHi isolates were collected from four different sites and clinical conditions such as otitis media, conjunctivitis, lower respiratory tract infection and normal oropharyngeal flora, whereas H. haemolyticus isolates were recovered from the oropharynx of healthy individuals.
These isolates were tested for invasion using the gentamicin survival assay with immortalised BEAS-2B (Sigma-Aldrich); isolated from normal human bronchial epithelium of non-cancerous individuals NHBE (Lonza); isolated from epithelial lining of airways above bifurcation of the lungs A549 (Public Health England); epithelial lung carcinoma cells derived from 58 year old Caucasian male and NCI-H292 (ATCC); muco-epidermoid pulmonary carcinoma cells derived from 32 year old female epithelia cell lines. Cell lines were grown and maintained in LHC8 (Gibco), BEGM (Lonza), DMEM growth medium (Sigma-Aldrich) supplemented with 2Mm Glutamine and 10% Foetal Bovine Serum (FBS), and RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS respectively at 370C in 5% CO$$_2$$.
Chapter 3 is a detailed study of the invasive ability of NTHi, possessing normal or PBP3, using four different and widely used respiratory cell types: BEAS-2B, NHBE, A549, and NCI-H292. The focus of this study was to evaluate if there is any difference in invasive ability of NTHi isolates individually, collectively and between isolates with normal and altered PBP3 across each cell type. The results showed that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the type of cell lines used and also confirmed the previous suggestions, provided by Okabe et al. (2010) and Atkins et al. (2014), that isolates with altered PBP3 possess more invasive ability compared to isolates with normal PBP3. Furthermore, the association between altered PBP3 and increased invasion was conserved across each cell line.
H.haemolyticus is considered to be a non-pathogenic commensal of the respiratory tract butlittle information is available on its ability to invade epithelial cells in vitro. If in vitro invasion is an indicator of ability for in vivo invasion and is important in the pathogenesis of NTHi infection, then H. haemolyticus would be expected to be comparatively non-invasive. As a result, Chapter 4 examined the invasive ability of H. haemolyticus to the BEAS-2B cell line. The invasion rate of 20 H. haemolyticus isolates were tested with BEAS-2B cell line and then 5/20 isolates were selected to test their invasion rate with other respiratory cell types used previously in this study. The results confirmed that non-invasiveness of H. haemolyticus isolates is consistent with their inability to cause respiratory infections.
In conclusion, this thesis has demonstrated the significant variability of invasion results across different epithelial cell lines, highlighting the importance of the choice of cell type in invasion assays as a confounding factor, as the ability of NTHi to invade respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the respiratory cell types. Furthermore, we have shown that the association between altered PBP3 and increased invasion is conserved across all the respiratory epithelial cell types used in this study. Finally, this thesis also revealed the inability of H. haemolyticus isolates to invade respiratory epithelial cell types in vitro, and suggests that this is consistent with their inability to cause opportunistic respiratory tract infections.

Item Type: Thesis - Research Master Singh, NK Nontypeable Haemophilus influenzae, Haemophilus haemolyticus, Invasion, Penicillin binding proteins, and Respiratory cell types Copyright 2017 the author Chapter 3 appears to be the equivalent of a post-print version of an article published as: Singh, N. K., Kunde, D. A., Tristram, S. G., 2016. Effect of epithelial cell type on in vitro invasion of nontypeable Haemophilus influenzae, Journal of microbiological methods, 129, 66-69 View statistics for this item