Neisseria meningitidis causes meningococcal disease but is frequently carried in the throats of healthy individuals; the factors that determine whether invasive disease develops are not completely understood. We carried out detailed studies of isolates, collected from patients and their household contacts, to identify differences between commensal throat isolates and those that caused invasive disease. Though isolates were identical by laboratory typing methods, we uncovered many differences in their genomes, in gene expression, and in their interactions with host cells. In particular, we found that several carriage isolates had lost their type IV pili, a surprising finding since pili are often described as essential for colonization. However, loss of type IV pili correlated with reduced secretion of a proinflammatory cytokine, TNF-α, when meningococci were cocultured with human bronchial epithelial cells; hence, the loss of pili could provide an advantage to meningococci, by resulting in a dampened localized host immune response.

<p>Neisseria meningitidis virulence is polygenic, therefore comparing many genomes may not yield strictly disease-associated virulence factors. An alternative approach is comparing closely related isolates, such as those from household contacts. Disease isolates have been shown to inhibit epithelial cell wound repair, while many carriage isolates do not. In this study, bacteria collected from disease patients and healthy household contacts were compared to identify the meningococcal factor responsible for wound repair inhibition and investigate how it contributes to invasive disease. Host cell wound repair inhibition was compared between disease-associated meningococcal isolate, NZ97/052, and isolates NZCM111 and NZCM112, from asymptomatic household contacts. Migrating bronchial airway cells were infected with meningococcal isolates and wound closure was evaluated by microscopy. NZ97/052 and NZCM111 both inhibited wound repair, whereas NZCM112 did not. To investigate if this was due to bacterial consumption of an important nutrient, infected cells were supplemented with nutrients known to be important for meningococcal growth and cell migration. Iron supplementation resulted in carriage associated isolates gaining the ability to inhibit wound repair. Genome and transcriptome comparisons were completed between NZ97/052 and NZCM112, which differ in wound repair inhibition. This analysis identified a frameshift mutation in NZCM112 in the haptoglobin-haemoglobin utilization gene, hpuA, which caused complete loss of expression. The hpuA gene was deleted by allelic replacement from NZ97/052, nullifying its ability to inhibit host cell migration. Furthermore, bacterial association and fluorescence microscopy assays suggested that HpuA contributes to meningococcal attachment to bronchial epithelial cells, as the hpuA mutant had significantly lower cell association. Heterologous expression of HpuA in E. coli resulted in higher levels of cell association, indicating that HpuA is sufficient to mediate bacterial adhesion to human bronchial epithelial cells. This work reveals novel roles for HpuA as a meningococcal adhesin and a bacterial factor that inhibits host cell migration.</p>

<p>Neisseria meningitidis virulence is polygenic, therefore comparing many genomes may not yield strictly disease-associated virulence factors. An alternative approach is comparing closely related isolates, such as those from household contacts. Disease isolates have been shown to inhibit epithelial cell wound repair, while many carriage isolates do not. In this study, bacteria collected from disease patients and healthy household contacts were compared to identify the meningococcal factor responsible for wound repair inhibition and investigate how it contributes to invasive disease. Host cell wound repair inhibition was compared between disease-associated meningococcal isolate, NZ97/052, and isolates NZCM111 and NZCM112, from asymptomatic household contacts. Migrating bronchial airway cells were infected with meningococcal isolates and wound closure was evaluated by microscopy. NZ97/052 and NZCM111 both inhibited wound repair, whereas NZCM112 did not. To investigate if this was due to bacterial consumption of an important nutrient, infected cells were supplemented with nutrients known to be important for meningococcal growth and cell migration. Iron supplementation resulted in carriage associated isolates gaining the ability to inhibit wound repair. Genome and transcriptome comparisons were completed between NZ97/052 and NZCM112, which differ in wound repair inhibition. This analysis identified a frameshift mutation in NZCM112 in the haptoglobin-haemoglobin utilization gene, hpuA, which caused complete loss of expression. The hpuA gene was deleted by allelic replacement from NZ97/052, nullifying its ability to inhibit host cell migration. Furthermore, bacterial association and fluorescence microscopy assays suggested that HpuA contributes to meningococcal attachment to bronchial epithelial cells, as the hpuA mutant had significantly lower cell association. Heterologous expression of HpuA in E. coli resulted in higher levels of cell association, indicating that HpuA is sufficient to mediate bacterial adhesion to human bronchial epithelial cells. This work reveals novel roles for HpuA as a meningococcal adhesin and a bacterial factor that inhibits host cell migration.</p>