Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier

Abstract

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

Figure 1.

Histological analysis of a case of meningococcal septic shock showing bacterial colonies in cerebral capillaries. Paraffin-embedded brain section samples were either analyzed by Gram-Weigert staining (A and B) or by immunohistochemistry with a polyclonal antibody directed against the strain cultured from the blood of this patient (C–F). (A) Brain section showing a bacterial microcolony inside a capillary. (B) Enlargement of the inset in A showing individual bacteria in the colony. (C) Two small colonies (arrowheads) visualized by immunohistochemistry. (D) Example of a large colony surrounded by edema. (E) Illustration of the favored localization of bacterial colonies in capillaries as opposed to arterioles. The arrowhead points to an arteriole and the arrow points to a connected capillary containing bacteria. (F) Enlargement of the inset in E. Arrowheads indicate immuno-labeled bacterial colonies present in the capillary.

Figure 2.

Analysis of N. meningitidis adhesion on endothelial cells in the presence of liquid flow using in a laminar flow chamber. (A) Endothelial cells forming a confluent monolayer on a glass slide are placed in the flow chamber and observed by phase contrast microscopy with a 20x lens. A typical sequence of three videoframes illustrates bacterial adhesion under various flow conditions: (B) before the introduction of bacteria into the chamber; (C) fluorescent bacteria flowing over the cells appear as short bright lines; and (D) bacteria adherent on the cell monolayer appear as still bright spots. Quantitative image analysis with the Quia software package allows automated identification of attached bacteria, which are then marked with colored squares. (E) Number of adherent bacteria determined as a function of time with the Quia software package shows bacteria accumulating with time in a linear fashion (circles). As a reference, results of a similar experiment performed in static conditions are also presented (squares). (F) The number of adherent bacteria plotted as a function of the concentration of bacteria introduced in the chamber shows a linear relationship between the inoculum and the number of adhering bacteria.

Figure 3.

Determination of the molecular basis of N. meningitidis adhesion under liquid flow and the optimal flow for efficient adhesion. (A) Testing of different mutants deficient for pili biogenesis and function shows that Tfp are necessary for adhesion under flow. After introduction in the flow chamber containing the endothelial cells in the presence of 0.04 dynes/cm², the number of bacteria per mm² was determined. Compared with the wild-type strain (WT), the pilE-deficient mutant (PilE) does not express the Tfp major subunit, the pilC1-deficient strain (PilC1) expresses nonfunctional pili, and the pilT mutant (PilT) fails to retract its pili. (B) The ability of N. meningitidis to adhere to an endothelial cell monolayer at different flow velocities was determined to show that increased shear stress strongly inhibits adhesion. The concentration of the bacterial suspension introduced in the chamber is constant throughout each experiment and only the shear stress levels varied. The number of bacteria per mm² is plotted as a function of shear stress level. Squares represent the results on HUVEC cells and circles on the brain-derived hCMEC/D3 endothelial cell line. (C) Comparison of adhesion behavior for three pilin variants at 0.04 (white bars) and 0.2 dynes/cm² (black bars) shows that adhesion is also inhibited by shear stress increase in these variants. Results are expressed as the percentage of the number of bacteria adhering at 0.04 dynes/cm².

Figure 4.

In vivo imaging of cerebral microcirculation in anesthetized rats equipped with a closed cranial window shows that local and transient blood flow reductions through cerebral capillaries provide conditions compatible with neisserial adhesion. A tracer dose of FITC-dextran was used to label the vascular network. Erythrocytes were labeled with FITC and injected into the animal. (A) Confocal microscope view through the cranial window of the superficial vascular network (10x lens) and a capillary network at a depth of ∼200 μm below the pia mater (20x lens) in the region corresponding to the inset in the left panel. The arrow points to a passing erythrocyte. (B) Example of tracking of erythrocytes using the Quia image treatment software to determine blood velocity in capillaries. Panels show the object detection and tracking at two successive time points separated by 400 ms. The arrow indicates flow direction. (C) Computed blood velocities in portions of two typical capillaries represented as a function of the position along the capillary (μ corresponds to the average velocity). (D) Blood velocities in a portion of capillary with temporary reduction in flow velocity (red line). (E) Blood velocities in another capillary exhibiting average low flow (63 μm/s) with a temporary further reduction in flow velocity (blue line). (F) Further analysis of the two periods of time where blood flow is reduced as described in D and E. Shear stress values were calculated from erythrocyte velocities and plotted as a function of time. The dotted line identifies shear stress levels compatible with neisserial adhesion (<0.5 dynes/cm²).

Figure 5.

After initial adhesion, N. meningititidis is resistant to high liquid flow and proliferates in the presence of high shear stress. (A) The ability of the wild-type strain to resist to high shear stress levels was determined. The number of adherent bacteria was first determined after adhesion at 0.04 dynes/cm² and washing of unbound bacteria at the same shear stress level. Shear stress was then gradually increased in 5-min steps up to 40 dynes/cm² and the number of bacteria that remained bound was determined. Results were plotted as a percentage of initial adhesion. (B) The ability of different strains to resist to high shear stress level was determined. After initial adhesion at 0.04 dynes/cm² (white bars) and shear stress increase at 3.5 dynes/cm² during 5 min, the number of bacteria that remained bound was determined (black bars). Results were plotted as a percentage of initial adhesion. Three different pilin variants (SB, SB*, and SA) and a pilus retraction mutant (PilT) were compared with wild type (WT). (C) Videoframes illustrating growth of adherent bacteria on cells under shear conditions (2 dynes/cm²). Bacteria were allowed to adhere at 0.04 dynes/cm², unbound bacteria were removed, and flow was increased and maintained at 2 dynes/cm² for the indicated times. Compact bacterial colonies appear and increase in size, reminding those observed in the sepsis case (Fig. 1).