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Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species
von Claudia LangeThe Gram-negative diplococci of the genus Neisseria comprises two obligate human pathogens:
N. meningitidis (Nme), a major cause of morbidity and mortality through septicaemia, haemorrhagical skin lesions and inflammation of the meninges, and N. gonorrhoeae (Ngo), the causative agent of the sexually transmitted disease gonorrhoea. Although both species exhibit distinct lifestyles and niche preferences in vivo, they share numerous virulence factors and immune evasion mechanisms, facilitating colonisation and survival in the human host. Type IV pili (Tfp) are the most important bacterial factors mediating initial attachment to human epithelial and endothelial cells, although the cellular interaction partner remains unknown. Outer membrane proteins such as Opa proteins are known to function as additional adhesins and invasins. The work presented here addressed two main questions regarding the molecular interplay between pathogenic Neisseria spp. and human epithelial and endothelial cells.
Firstly, it was investigated whether Nme crosses endothelial cell monolayers, such as the bloodbrain barrier, paracellularly or transcellularly, and which bacterial factor is necessary for its traversal. It was also questioned if Ngo could follow a similar route of transition. A novel in vitro model comprising primary human umbilical vein endothelial cells and the automated cellmonitoring system CellZscope®, was established to determine changes in the barrier stability of polarised endothelial cell monolayers upon infection in real-time. Infection by various Nme and Ngo strains resulted in a highly significant transient loss of the barrier integrity with strain-specific differences. This observation demonstrated a disruption of cell-cell contacts allowing for a paracellular passage. Nme and Ngo mutant strains featuring an unpiliated phenotype also caused a loss of barrier integrity, indicating that additional bacterial factor(s) were involved in the process. Moreover, an Opa protein ‘switch on’ was observed for phenotypically Opa negative selected Ngo strains. That could be correlated to a delayed onset of barrier disruption, compared to Opa protein expressing Ngo. Furthermore, infection by an unpiliated Ngo deletion mutant devoid of all opa genes resulted in significantly reduced effects, indicating an important role for Opa proteins. However, a piliated variant of the same mutant was again able to cause a transient barrier disruption similar to other strains previously tested. These results led to the conclusion that Tfp and Opa proteins might function redundantly. Concludingly, Nme and Ngo seem to be equally capable to cross endothelial cell barriers via a paracellular route, dependent on the presence of either Tfp or Opa proteins.
Secondly, an unbiased RNAi based screening approach in combination with automated fluorescent microscopy and image analysis was set up to elucidate the identity of the cellular pilus receptor and additional factors involved in the infection process constituting microcolony (MC) and subsequent cortical plaque formation. Five parameters of infection were analysed upon specific gene knockdown: (a) Tfp mediated adherence of Ngo, (b) number of MC relative to cell number, (c) proportion big MC to medium size MC, (d) actual size of MC and (e) actin recruitment underneath MC. The epithelial reporter cell line ME-180ActRFP03 was generated allowing for direct, high-quality visualization of the cellular actin cytoskeleton. Conducting two sequential screening rounds of a defined siRNA library, 122 target genes were identified. Subsequent enrichment analysis for canonical signalling pathways confirmed the actin cytoskeleton signalling, signal transduction pathways and central intracellular signalling networks to be involved in the Ngo infection process. Three major branches of the actin cytoskeleton signalling network regulating actin polymersiation and stabilisation, actomyosin contractibilty and focal adhesion assembly, were determined to be engaged by Ngo. Moreover, multiparameter profile analysis identified targets essential for actin recruitment to MC and MC formation. Furthermore, receptor tyrosine kinases belonging to the ephrin receptor, the ErbB receptor, the FGF receptor and the DDR subfamilies were found to play a role during Ngo attachment, MC formation and Ngo induced actin recruitment. In sum, these functional studies demonstrated that the successful establishment of an Ngo infection on epithelial cells depends on a complex pathogen-host cell interplay defined by mutual signalling processes.
N. meningitidis (Nme), a major cause of morbidity and mortality through septicaemia, haemorrhagical skin lesions and inflammation of the meninges, and N. gonorrhoeae (Ngo), the causative agent of the sexually transmitted disease gonorrhoea. Although both species exhibit distinct lifestyles and niche preferences in vivo, they share numerous virulence factors and immune evasion mechanisms, facilitating colonisation and survival in the human host. Type IV pili (Tfp) are the most important bacterial factors mediating initial attachment to human epithelial and endothelial cells, although the cellular interaction partner remains unknown. Outer membrane proteins such as Opa proteins are known to function as additional adhesins and invasins. The work presented here addressed two main questions regarding the molecular interplay between pathogenic Neisseria spp. and human epithelial and endothelial cells.
Firstly, it was investigated whether Nme crosses endothelial cell monolayers, such as the bloodbrain barrier, paracellularly or transcellularly, and which bacterial factor is necessary for its traversal. It was also questioned if Ngo could follow a similar route of transition. A novel in vitro model comprising primary human umbilical vein endothelial cells and the automated cellmonitoring system CellZscope®, was established to determine changes in the barrier stability of polarised endothelial cell monolayers upon infection in real-time. Infection by various Nme and Ngo strains resulted in a highly significant transient loss of the barrier integrity with strain-specific differences. This observation demonstrated a disruption of cell-cell contacts allowing for a paracellular passage. Nme and Ngo mutant strains featuring an unpiliated phenotype also caused a loss of barrier integrity, indicating that additional bacterial factor(s) were involved in the process. Moreover, an Opa protein ‘switch on’ was observed for phenotypically Opa negative selected Ngo strains. That could be correlated to a delayed onset of barrier disruption, compared to Opa protein expressing Ngo. Furthermore, infection by an unpiliated Ngo deletion mutant devoid of all opa genes resulted in significantly reduced effects, indicating an important role for Opa proteins. However, a piliated variant of the same mutant was again able to cause a transient barrier disruption similar to other strains previously tested. These results led to the conclusion that Tfp and Opa proteins might function redundantly. Concludingly, Nme and Ngo seem to be equally capable to cross endothelial cell barriers via a paracellular route, dependent on the presence of either Tfp or Opa proteins.
Secondly, an unbiased RNAi based screening approach in combination with automated fluorescent microscopy and image analysis was set up to elucidate the identity of the cellular pilus receptor and additional factors involved in the infection process constituting microcolony (MC) and subsequent cortical plaque formation. Five parameters of infection were analysed upon specific gene knockdown: (a) Tfp mediated adherence of Ngo, (b) number of MC relative to cell number, (c) proportion big MC to medium size MC, (d) actual size of MC and (e) actin recruitment underneath MC. The epithelial reporter cell line ME-180ActRFP03 was generated allowing for direct, high-quality visualization of the cellular actin cytoskeleton. Conducting two sequential screening rounds of a defined siRNA library, 122 target genes were identified. Subsequent enrichment analysis for canonical signalling pathways confirmed the actin cytoskeleton signalling, signal transduction pathways and central intracellular signalling networks to be involved in the Ngo infection process. Three major branches of the actin cytoskeleton signalling network regulating actin polymersiation and stabilisation, actomyosin contractibilty and focal adhesion assembly, were determined to be engaged by Ngo. Moreover, multiparameter profile analysis identified targets essential for actin recruitment to MC and MC formation. Furthermore, receptor tyrosine kinases belonging to the ephrin receptor, the ErbB receptor, the FGF receptor and the DDR subfamilies were found to play a role during Ngo attachment, MC formation and Ngo induced actin recruitment. In sum, these functional studies demonstrated that the successful establishment of an Ngo infection on epithelial cells depends on a complex pathogen-host cell interplay defined by mutual signalling processes.