The type IV pili (Tfp) of pathogenic Neisseria (i.e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized. In this work, we analyzed bacterial motility and monitored Tfp retraction using live-cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 μm/s, whereas N. gonorrhoeae moved with a lower speed (1.0 μm/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the −10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed. These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.
The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.
ABSTRACT Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis .
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