Difference in virulence between Neisseria meningitidis serogroups W and Y in transgenic mice

Abstract

Background: Neisseria meningitidis serogroups W and Y are the most common serogroups causing invasive meningococcal disease in Sweden. The majority of cases are caused by the serogroup W UK 2013 strain of clonal complex (cc) 11, and subtype 1 of the serogroup Y, YI strain of cc23. In this study, virulence factors of several lineages within cc11 and cc23 were investigated in transgenic BALB/c mice expressing human transferrin. Transgenic mice were infected intraperitoneally with serogroup W and Y isolates. Levels of bacteria and the proinflammatory cytokine CXCL1 were determined in blood collected 3 h and 24 h post-infection. Apoptosis was investigated in immune cells from peritoneal washes of infected mice. Adhesion and induction of apoptosis in human epithelial cells were also scored.

Results: The levels of bacteraemia, CXCL1, and apoptosis were higher in serogroup W infected mice than in serogroup Y infected mice. Serogroup W isolates also induced higher levels of apoptosis and adhesion in human epithelial cells. No significant differences were observed between different lineages within cc11 and cc23.

Conclusions: N. meningitidis Serogroup W displayed a higher virulence in vivo in transgenic mice, compared to serogroup Y. This was reflected by higher bacteremia, proinflammatory activity, and ability to induce apoptosis in mouse immune cells and human epithelial cells.

Keywords: Neisseria meningitidis; Serogroup W; Serogroup Y; Transgenic mice; Virulence.

Fig. 1

Temperature variation in mice infected with Neisseria meningitidis serogroup W and Y isolates. Temperature was measured transcutaneously using an infrared device 3 h and 24 h post- infection and expressed as differences in °C from the temperature before infection for each individual mouse in each group as indicated. The mean differences and standard errors are shown for each group. *** indicates the level of the significant difference in temperature between mice infected with serogroup W isolates compared to serogroup Y infected mice (p < 0.0001). * indicates the level of the significant difference between mice infected with the invasive serogroup W UK 2013 strain compared to mice infected with the UK 2013 strain isolates from the nasopharynx/throat (p < 0.01)

Fig. 2

a: Amount of bacteria (CFU/mL) in blood 3 h and 24 h post-infection with N. meningitidis serogroup W and Y isolates. The bars represent the median and 95% confidence interval. Differences between and within serogroups were compared using a Mann-Whitney test. *** indicates the level of the significant difference between serogroup W infected mice compared to serogroup Y infected mice (p < 0.0001). b: Amount of the proinflammatory cytokine CXCL1 in ng/mL detected in blood of N. meningitidis serogroup W and Y infected mice after 3 h and 24 h. The bars represent the median and 95% confidence interval. Differences between and within serogroups were compared using a Mann-Whitney test. *** indicates the level of the significant difference between serogroup W infected mice compared to serogroup Y infected mice (p < 0.0001)

Fig. 3

Histogram overlays of apoptosis of immune cells in the peritoneal washes of infected mice. The apoptosis is displayed as fluorescence levels on the x-axis. Three isolates represent each lineageof the N. meningitidis serogroups. The data from infected mice were compared to data from two non-infected mice as controls. Serogroup W isolates are displayed in histogram a: non-infected mice (grey), original UK isolate (red), invasive 2013 strain isolate (blue), and nasopharynx/throat (NP) 2013 strain isolate (green). Serogroup Y isolates are displayed in histogram b: YI subtype 1 (blue), YI subtype 2 (green), cc23 other (red), and non-infected mice (grey). The percentages of cells in apoptosis and standard errors are displayed for each isolate as indicated by the corresponding colours

Fig. 4

Staining of immune cells in peritoneal wash from one representative N. meningitidis serogroup W (isolate PubMLST ID 57576) infected mouse, 3 h post-infection compared to a non-infected mouse. The defined immune cells are gated on the forward (FSC) and scattered (SSC) axis and are seen in red. These cells were then observed according to the specific phycoerythrin (PE) fluorochrome staining (FL2 axis) for macrophages (F4/80+), monocytes (Ly6C+), and neutrophils (Ly6G+), represented in black for both infected and non-infected mice. In the infected mouse, these cells were gated for apoptosis using FITC-labelled Annexin V (FL1 axis), which is represented by green colour. The percentage was calculated for gated cells from all the observed cells, which is indicated in each gate as well as the names of gated cells

Fig. 5

Percentage of apoptosis in Hec-1-B cells 3 h post-infection with serogroup N. meningitidis W and Y isolates. Lines represent the median and 95% confidence intervals. The comparison between and within serogroups was performed using a Mann-Whitney test. *** indicates the significant difference in apoptosis between mice infected with serogroup W isolates compared to serogroup Y infected mice (p<0.0001)

Fig. 6

DAPI staining of Hec-1-B cells. a: Hec-1-B cells infected with a N. meningitidis serogroup Y isolate without adherent bacteria, b: Hec-1-B cells infected with a N. meningitidis serogroup W isolate with adherent bacteria as revealed by DAPI (which stains nucleic acids)

Fig. 7

Comparison of number of bacteria adherent to cells 3 h post-infection. Lines represent the mean and 95% confidence intervals. A Mann-Whitney test was used to compare serogroups W and Y. *** indicates the level of the significant difference in adhesion between mice infected with serogroup W isolates compared to serogroup Y infected mice (p = 0.0004)

Fig. 8

a: Western blot of IgA1 protease in a collection of serogroup W and Y isolates and compared to a serogroup B isolate (MC58). A mutant strain, Δiga, was used as a negative control [6]. The IgA1 protease consisting of the IgaP and Igaα domains generates a protein of size 150 kDa in MC58. An IgA1 protease consisting of only the IgaP domain results in a protein with a size of 100 kDa in MC58. The serogroup W isolates generated a protein that was smaller than 150 kDa. b: PCR amplified Igaα domains of serogroup W and Y isolates compared to MC58, which has a complete Igaα domain. The complete Igaα domain of MC58 has a size of approximately 1.5 kb while the serogroup W and Y isolates are smaller, approximately 0.9 kb. The serogroup Y isolates seem to have a slightly smaller Igaα domain than serogroup W