Peripheral blood vessels are a niche for blood-borne meningococci

Abstract

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.

Keywords: Neisseria meningitidis; Tn-seq; host cell interaction; nutritional virulence; purpura fulminans.

Figure 1.

Neisseria meningitidis colonization of the human skin graft leads to mice lethality. (A) Kaplan-Meier plot showing the survival of grafted (n = 8; dot) and non-grafted (n = 8; square) mice after intravenous challenge with 5.10⁶ CFU of N. meningitidis wt 2C4.3 strain (p-value = 0,003; Log-rank Mantel-Cox survival analysis) or grafted mice infected with 5.10⁶ CFU of N. meningitidis non-adhesive PilC1 defective mutant (n = 6; triangle). (B) Transversal human skin graft sections after infection by N. meningitidis wt 2C4.3 strain and the PilC1 non-adhesive defective derivative (bacteria, red) associated with human vessels (collagen IV-basal lamina, green) in the human grafted skin 4 hrs after infection. (C) Blood and graft bacterial loads of grafted and non-grafted SCID mice infected intravenously with the wt N. meningitidis 2C4.3 strain. Bacterial counts are expressed in CFU/ml for blood and in CFU/g for human skin grafts (non-grafted mice, n = 7; grafted mice, n = 5). Horizontal lines represent the mean bacterial load. Statistical significance was determined using the Wilcoxon-Mann-Whitney U-test (p = 0.0025). (D) Total bacterial load of grafted and non-grafted SCID mice shown in panel B (see material and methods section). Bacterial counts are expressed in CFU/mice for the inoculum and 18 hrs after infection (non-grafted mice, n = 7; grafted mice, n = 5). Horizontal lines represent the mean bacterial load. Statistical significance was determined using One-Way ANOVA (**: p<0.01). (E) Blood bacterial loads of grafted SCID mice infected intravenously with the PilC1 defective N. meningitidis 2C4.3 strain (ΔPilC1). Bacterial counts are expressed in CFU/ml of blood. Horizontal lines represent the mean bacterial load. P-value was determined using the Wilcoxon-Mann-Whitney U-test.

Figure 2.

Tn-seq analysis. (A) Distribution of the CDS containing random Tn insertions on the genome map of N. meningitidis Z2491. Outer circles: Green bars indicate the N. meningitidis Z2491 CDS. Inner circles: Red and black bars indicate the essential/growth-defective and non-growth defective gene-disrupted mutants, respectively, in broth; turquoise and purple bars indicate the gene-disrupted mutants with a significant fitness change in the blood of non-grafted mice 4 hrs and 18 hrs after infection; dark blue and pink bars indicate the gene-disrupted mutants with a significant fitness change in the human skin grafts of grafted mice 4 hrs and 18 hrs after infection. (B) Venn diagram representing the absolute number of gene-disrupted mutants with a significant differential fitness decrease (log₂FC< −2 with a p-value<0.05) in any of the four tested conditions: blood from non-grafted mice 4 hrs and 18 hrs after infection, human skin grafts 4 hrs and 18 hrs after infection.

Figure 3.

In vivo competition assay of defective mutants in blood of non-grafted mice 18 hrs after infection. A mixture of the N. meningitidis wt Z5463 strain and one of the 3 different mutant (ΔputP, ΔcysG, ΔNMA0486) displaying a mild fitness decrease for survival in the blood of non-grafted mice 18 hrs infection in our Tn-seq screening were tested for competition in vivo in Balb/C mice. A mutant derivative strain not having a fitness change in vivo (ΔpilC2) was used as a control. Competition indexes confirmed the Tn-seq fitness predictions. The 3 defective mutants were out-competed by the wt parental strain 18 hrs after infection, which was not the case of the neutral mutant ΔpilC2. Statistical significance was determined for each mutant using One-Way ANOVA (*: p < 0.05; **: p < 0.01; ****: p < 0.0001).