The role of heparin binding haemagglutinin adhesin and curli pili on the pathogenicity of Mycobacterium tuberculosis.
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Background: Phagocytic host cells drive both the innate and adaptive arms of the host immune response during Mycobacterium tuberculosis (M. tuberculosis) infection. M. tuberculosis modulates the host immune responses and is able to proliferate in macrophages. The structures that mediate M. tuberculosis adherence (Adhesins) to macrophages are of particular interest for therapeutic development due to their cell surface localisation and immunogenic characteristics. M. tuberculosis produces numerous antigens that display adhesin functionality, including heparin-binding haemagglutinin adhesin (HBHA) and M. tuberculosis curli pili (MTP) that are critical for adherence to host cells. Recently, the independent elucidation of the immunogenic potential of each suggested that HBHA and MTP may represent a novel combination as a biomarker for future therapeutic development. This study aimed to elucidate the effect of HBHA and MTP in combination on adhesion, invasion, replication, cytokine production and transcription regulation of macrophages infected with HBHA and MTP proficient and deficient strains in an attempt to assess their immunogenic capacity. Materials and methods: THP-1 monocytic cells were differentiated into macrophages and infected at a multiplicity of infection of 5 with single mutants (ΔhbhA and Δmtp), single complements of double mutant (hbhA comp and mtp comp), MTP and HBHA deficient double mutant ΔhbhA-mtp (DM) and MTP and HBHA proficient wild-type (WT) strain. The relative percentage adhesion/ invasion of the mutant and complemented strains was calculated at 1 h and 2 h post-infection respectively and compared to wild-type. Intracellular replication was quantified by colony forming units at 4 h, day 3 and day 6 post-infection. To assess host transcriptomic changes elicited during early infection of THP-1 differentiated macrophages by WT and DM, RNA was extracted from host cells at 4 h post-infection. For the biological adhesion data set, raw data were filtered for genes in common with the Gene Ontology biological adhesion dataset sourced from EntrezGeneIds using the molecular signatures database with a False Discovery Rate q-value <1 (Chapter 1). Significantly differentially expressed genes with a p value <0.05 were used for further enrichment analysis (Chapter 2 and 3). Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, USA) upstream regulator, canonical pathway and biofunctions enrichment analysis were used to further investigate the differential regulation of molecular signatures by MTP and HBHA proficient and deficient strains. Macrophage cytokine/chemokine production was quantified at 24, 48 and 72 h post-infection using the Bio-Plex Pro Human Cytokine Multi-Plex Panel (Bio-Rad). Real-time quantitative RT-PCR was used to validate RNA sequencing findings and investigate transcriptional regulation of HBHA and MTP of following genes: CD80, DLX3, NLRP3, TGM5 and TLR2 at 1 h, 2 h and 4 h post-infection Results: During adhesion, DM induced a similar decrease in percentage adhesion (33.16%) to Δmtp (39.4%), ΔhbhA (22.78%), mtp comp (24.72%), but statistically lower decrease in percentage adhesion than hbhA comp (53.85%). During invasion, DM displayed a significant decrease in percentage invasion (36.49%) compared to Δmtp (61.49%) and hbhA comp (53.85%); and significantly higher decrease in percentage invasion than ΔhbhA (22.29%) and mtp comp (24.72%). Δmtp demonstrated a 39.4% and 61.49% decrease in percentage adhesion and invasion compared to WT respectively. The HBHA-MTP proficient strain induced greater transcriptional changes resulting in enhanced adhesion to phagocytes and invasion of cells. Furthermore, the HBHA-MTP proficient strain displayed the sole ability to induce activation of phagocytosis. Further investigation of canonical pathway differential regulation by HBHA-MTP proficient strain demonstrated greater induction of canonical pathways. The most differentially regulated pathway was Gαq signalling canonical pathway, which is vital for migration of phagocytes. In addition, the HBHA-MTP proficient strain also enhanced activation of the acute phase response, role of pattern recognition receptors in recognition of bacteria and viruses, and production of nitric oxide and reactive oxygen species in macrophages canonical pathways. RNA sequencing analysis showed that the M. tuberculosis adhesins, HBHA and MTP, elicited differential transcriptional regulation in macrophages, and demonstrated that predicted upstream regulators were associated with cytokine production. Further investigation of canonical pathways associated with these upstream regulators and cytokine quantification revealed that HBHA and MTP activate NF-κB, toll-like receptor, p38 MAPK and PI3-K/AKT canonical signalling pathways. HBHA and MTP elicited greater production of IL-4 and IL-10 at 24 h; G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-10, IL-12, IL-17, IFN-γ and TNF-α at 48 h and G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IFN-γ and TNF-α at 72 h respectively, compared to DM infection. IL-1β, IL-2, IL-6, IL-12(p70), IL-17, TNF-α, IFN-γ, colony-stimulating factors G-CSF, GM-CSF and chemokines MCP-1 and MIP-1β were produced in higher concentrations by M. tuberculosis infection than anti-inflammatory cytokines IL-4, IL-5, IL-10 and IL-13. The bacillary load of M was significantly less than WT at all time intervals and similar to DM. The decreased replication ability of the HBHA-MTP mutant was attributed to MTP and not HBHA, suggesting that MTP facilitates replication during infection of macrophages. A transcriptional response common to both WT and DM, independent of HBHA-MTP, as well as unique responses induced by HBHA-MTP presence and deficiency were observed. The common transcriptional pattern exhibited the most enrichment for granulocyte adhesion and diapedesis canonical pathway, TNF upstream regulation and migration of cells biological function. The HBHA-MTP uniquely induced transcripts were associated with the most significant enrichment of the Adipogenesis pathway, whilst HBHA-MTP deficiency induced the most significant enrichment of T helper cell differentiation. Unique transcripts elicited by HBHA-MTP deficiency induced less enrichment of NF-κB upstream regulator and were associated with migration of cells. The top 10 canonical pathways enriched by all transcripts were similar between both infections, but differed in molecules involved and their significance. HBHA-MTP enriched the TREM1 signalling pathway to a greater degree than HBHA-MTP deficiency in macrophages. HBHA-MTP deficiency, but not presence, enriched Th1 and Th2 Activation, Th1, Th2, Melatonin degradation, Sumoylation, Methylglyoxal degradation III, Granzyme A signalling, PCP pathways. Discussion and conclusion: MTP played a greater role in adhesion and invasion during independent knockout and complementation in the double knockout strain than HBHA. HBHA and MTP together induced transcriptional changes that favour adhesion and invasion of macrophages. In addition, these 2 adhesins serve as pathogen-associated molecular patterns that enable host immune recognition during early infection of macrophages. HBHA and MTP activate intracellular signalling pathways that result in the longitudinal enhancement of a pro-inflammatory response during M. tuberculosis infection of macrophages. HBHA and MTP predominately induced a pro-inflammatory cytokine profile instead of an anti-inflammatory cytokine profile. This suggests that HBHA and MTP play a role in protective immunity and immunopathology as a consequence of pro-inflammatory cytokines such as TNF-α and minimal anti-inflammatory cytokines during M. tuberculosis infection. HBHA and MTP deficiency led to advanced immune activation and decreased intracellular growth. This suggests in the absence of HBHA and MTP, the presence of multiple, alternate antigens stimulate the intracellular signalling and transcriptional regulation in vitro. This advanced immune activation would potentially be detrimental to M. tuberculosis establishing a successful infection and would suggest that HBHA and MTP play a role in host immune response modulation as a protective measure during initial infection. Further investigation into the identity of these antigens would possibly result in a more successful, novel therapeutic target combination in addition to HBHA and MTP.