The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

Peripheral blood vessels are a niche for blood-borne meningococci.

Neisseria meningitidis is the causative agent of cerebrospinal meningitis and that of a rapidly progressing fatal septic shock known as purpura fulminans. Meningococcemia is characterized by bacterial adhesion to human endothelial cells of the microvessels. Host specificity has hampered studies on the role of blood vessels colonization in N. meningitidis associated pathogenesis. In this work, using a humanized model of SCID mice allowing the study of bacterial adhesion to human cells in an in vivo context we demonstrate that meningococcal colonization of human blood vessels is a prerequisite to the establishment of sepsis and lethality. To identify the molecular pathways involved in bacterial virulence, we performed transposon insertion site sequencing (Tn-seq) in vivo. Our results demonstrate that 36% of the genes that are important for growth in the blood of mice are dispensable when bacteria colonize human blood vessels, suggesting that human endothelial cells lining the blood vessels are feeding niches for N. meningitidis in vivo. Altogether, our work proposes a new paradigm for meningococcal virulence in which colonization of blood vessels is associated with metabolic adaptation and sustained bacteremia responsible for sepsis and subsequent lethality.

Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization.

UNLABELLED: Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. IMPORTANCE: Meningococcal meningitis is a common cause of meningitis in infants and adults. Neisseria meningitidis (meningococcus) is also a commensal bacterium of the nasopharynx and is carried by 3 to 30% of healthy humans. Under some unknown circumstances, N. meningitidis is able to invade the bloodstream and cause either meningitis or a fatal septicemia known as purpura fulminans. The onset of symptoms is sudden, and death can follow within hours. Although many meningococcal virulence factors have been identified, the mechanisms that allow the bacterium to switch from the commensal to pathogen state remain unknown. Therefore, we used a Tn-seq strategy coupled to high-throughput DNA sequencing technologies to find genes for proteins used by N. meningitidis to specifically colonize epithelial cells and primary brain endothelial cells. We identified 383 genes and 8 intergenic regions containing sRNAs essential for growth and 288 genes and 33 intergenic regions containing sRNAs required specifically for host cell colonization.

Epistasis as a determinant of the HIV-1 protease's robustness to mutation.

The robustness of phenotypes to mutation is critical to protein evolution; robustness may be an adaptive trait if it promotes evolution. We hypothesised that native proteins subjected to natural selection in vivo should be more robust than proteins generated in vitro in the absence of natural selection. We compared the mutational robustness of two human immunodeficiency virus type 1 (HIV-1) proteases with comparable catalytic efficiencies, one isolated from an infected individual and the second generated in vitro via random mutagenesis. Single mutations in the protease (82 and 60 in the wild-type and mutant backgrounds, respectively) were randomly generated in vitro and the catalytic efficiency of each mutant was determined. No differences were observed between these two protease variants when lethal, neutral, and deleterious mutations were compared (P = 0.8025, chi-squared test). Similarly, average catalytic efficiency (-72.6% and -64.5%, respectively) did not significantly differ between protease mutant libraries (P = 0.3414, Mann Whitney test). Overall, the two parental proteins displayed similar mutational robustness. Importantly, strong and widespread epistatic interactions were observed when the effect of the same mutation was compared in both proteases, suggesting that epistasis can be a key determinant of the robustness displayed by the in vitro generated protease.

Significant changes in integrase-associated HIV-1 replication capacity between early and late isolates.

During the human immunodeficiency virus type 1 (HIV-1) pandemic, continuous, extensive genetic diversification of the virus has been observed. To study the effect of HIV-1 diversification on integrase-associated viral replication capacity (RC), 94 HIV-1 subtype B integrase sequences from two groups of antiretroviral-naive viruses isolated 15 y apart were amplified and recombined with an HIV-1 infectious clone. Viral RC was determined by infecting a T cell line with a long terminal repeat-driven green fluorescent protein reporter. Significant differences in integrase-mediated RC were observed between recombinant viruses from early and late isolates (p=0.0286). Integrases from late isolates had significantly lower sequence conservation scores compared to an ancestral subtype B sequence (p<0.0001). Integrase amino acid polymorphisms S17N, I72V, S119P, and D256E were associated with a lower ex vivo viral RC. These results suggest that integrase sequence diversification has affected ex vivo HIV-1 RC.

Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness.

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.