Inactivation of the porB gene reduces the virulence of Neisseria meningitidis in transgenic mice

Abstract

Background: Neisseria meningitidis is a human pathogen, carried asymptomatically in the nasopharynx, that can also cause invasive meningococcal disease. Understanding the carriage/invasiveness balance is crucial, and bacterial genetic factors may impact this balance. A previous genome-wide association study reported that the gene porB class 3 was significantly associated with carriage isolates. This study aimed to examine the impact of porB variants on virulence in carriage and invasive meningococcal isolates.

Results: For this, 24 isolates were used (13 invasive and 11 carriage) belonging to different genogroups (B, C, W, Y, and cnl) and selected based on the presence of the genetic variant porB class 2 or class 3. Transgenic BALB/c mice expressing human transferrin were infected intraperitoneally with these isolates. After 3 and 24 h of infection, clinical scores (fur quality, strength, and temperature) and bacterial load in blood were used to evaluate bacterial virulence. The concentrations of inflammatory cytokines were determined from blood. PorB^- mutants were created from a carriage and an invasive isolate, and were tested in transgenic mice. The invasive isolates provoked significantly more severe infections compared to the carriage isolates, and the carriage isolates of porB class 3 were significantly less virulent than the invasive isolates of porB class 2 or 3. The invasive PorB^- isolate caused milder infections than the parental isolate.

Conclusions: This study confirms the ability of invasive isolates of N. meningitidis to cause more severe infections than carriage isolates in transgenic mice. The porB expressed in invasive isolates seems to contribute to their higher virulence compared to carriage isolates, although this effect may depend on the genomic context. Notably, differences in virulence were mainly observed among serogroup C and W isolates.

Keywords: Neisseria meningitidis; PorB; Carriage; Experimental infection; Inflammatory cytokines; Invasive meningococcal disease; Transgenic mice.

Fig. 1

Temperature variation in mice infected with carriage and invasive isolates of Neisseria meningitidis, at 3 h and 24 h post-infection from each experiment. Non-infected mice are also included. The bars represent medians. Statistical significance was calculated using the Mann-Whitney U-test. At 24 h,the mice infected with invasive isolates showed significantly lower temperature than the mice infected with carriage isolates (p < 0.05). The dotted oval at 24 h highlights isolates of serogroups C and W. Data on capsule expression are shown in Table 1

Fig. 2

Bacterial load in mice infected with carriage and invasive isolates of Neisseria meningitidis at the time of infection (0 h; representing the inoculum) and at 3 h and 24 h post-infection from each experiment. The bacterial load at 0 h reflects the initial concentration of the bacterial suspension used for intraperitoneal injection and not the bacterial load in blood. Bars represent medians. Statistical significance was calculated using the Mann-Whitney U-test. The mice infected with invasive isolates showed a significantly higher number of bacteria than the mice infected with carriage isolates at both 3 h (p < 0.01) and 24 h (p < 0.001). The dotted oval at 24 h highlights isolates of serogroups C and W. Data on capsule expression are shown in Table 1

Fig. 3

Concentrations of cytokines 3 h and 24 h post-infection in mice infected with carriage and invasive isolates of Neisseria meningitidis, and in non-infected mice from each experiment. Results are expressed as medians. Statistical significance was calculated using the Mann-Whitney U-test. (A) TNF-α levels were significantly higher in the mice infected with invasive isolates at 3 h (p < 0.01) and 24 h (p < 0.05). (B) IL-6 levels were significantly higher in the mice infected with the invasive isolates at 3 h (p < 0.05) and 24 h (p < 0.01). (C) CXCL1/KC levels were significantly higher in the mice infected with the invasive isolates at 3 h (p < 0.05) and 24 h (p < 0.01)

Fig. 4

Temperature variation in mice infected with the parental isolates McBar-21 (carriage) or 19–977 (invasive) and their respective porB mutants CK5 and CK8,at 3 h and 24 h post-infection. Bars represent medians. Statistical significance was calculated using the Mann-Whitney U-test. Non-infected mice are also included. (A) Mice infected with the parental carriage isolate McBar-21 and those infected with its porB mutant CK5 showed no significant differences. (B) Mice infected with the parental invasive isolate 19–977 showed a significantly lower temperature than those infected with its porBmutant CK8 at 24 h (p < 0.0001)

Fig. 5

Bacterial load in mice infected with the parental isolates McBar-21 (carriage) or 19–977 (invasive) and their respective porBmutants CK5 and CK8, at the time of infection (0 h; representing the inoculum) and at 3 h and 24 h post-infection. The bacterial load at 0 h reflects the initial concentration of the bacterial suspension used for intraperitoneal injection and not the bacterial load in blood. Bars represent medians. Statistical significance was calculated using the Mann-Whitney U-test. (A) Mice infected with the parental carriage isolate Mcbar-21 and those infected with its porB mutant CK5 showed no significant differences. (B) Mice infected with the parental invasive isolate 19–977 showed a significantly higher number of bacteria than those infected with its porB mutant CK8 at 24 h (p < 0.0001).

Fig. 6

Concentrations of cytokines 3 h and 24 h post-infection in mice infected with the parental invasive isolate 19–977 and its porB mutant CK8 and in non-infected mice from each experiment. Bars represent medians. Statistical significance was calculated using the Mann-Whitney U-test. (A) TNF-α levels were significantly higher in the mice infected with the parental isolate at 3 h (p < 0.0001), with no significant difference at 24 h. (B) IL-6 levels were significantly higher in the mice infected with the parental isolate at 24 h (p < 0.0001),with no significant difference at 3 h. (C) CXCL1/KC levels were significantly higher in the mice infected with the parental isolate at 24 h (p < 0.0001), with no significant difference at 3 h