Gene regulation in Streptococcus pneumoniae - RePub - Erasmus ...

The CodY regulon of S. pneumoniae
washed with 70% ethanol, air dried, and resuspended in 10 µl formamide loading buffer
(containing bromophenol-blue and xylene-blue). These samples were heated for 1 min at
99ºC, and applied to a preheated 8% polyacrylamide denaturing sequencing gel. Gels were
air-dried, and autoradiography was performed by exposing the gel to an X-ray film.
In vitro pneumococcal adherence assay
Adherence of pneumococci to epithelial cells was studied essentially as described
previously (7, 27, 28). In short, monolayers of the human pharyngeal cell line Detroit 562
(ATCC CCL-138) were washed twice with 1 ml PBS. Aliquots of bacteria (grown to mid-log
in Todd Hewitt Yeast-broth) stored at -80°C were thawed rapidly, harvested by
centrifugation, and resuspended in RPMI 1640 medium without phenol red (Invitrogen)
supplemented with 1% FCS to 1x10 7 CFU/ml. One ml of bacterial suspension was allowed to
adhere for 2h, after which non-adherent bacteria were removed by three washes with 1 ml
PBS. For quantification of adherence, epithelial cells were subsequently detached by
treatment with 25% Trypsin, 1 mM EDTA in PBS and lysed by the addition of ice-cold
0.025% Triton X-100 in PBS. Serial 10-fold dilutions were plated on blood agar plates to
count the number of adherent bacteria, and corrected mathematically to account for small
differences in count in the initial inoculum. Wild-type and mutant strains grew comparably in
RMPI medium without Detroit 562 cells.
Experimental mouse models
Nine-week old female outbred CD-1 mice (Harlan, Horst, Netherlands) were used for
all infection models. Prior to infection, D39 wild-type and ΔcodY were passaged in mice to
maintain virulence as described previously (25). Cultures of S. pneumoniae D39 or ΔcodY
were grown to an OD600 of 0.3, and stored in aliquots at -80�C in 10% glycerol. Prior to
infection, these aliquots were spun down and bacteria were resuspended in sterile PBS. Mice
were lightly anesthetized with 2.5% (vol/vol) isoflurane / O2, and infected intranasally with
10 6 CFU of bacteria as described previously (24). At predetermined time points after
infection, groups of mice were sacrificed by cervical dislocation and samples of various sites
were taken to determine the bacterial load. In the colonization model, five mice per group
were infected with 10 µl of PBS containing 10 6 CFU of either D39 wild-type or ΔcodY, a
volume small enough to only infect the nose (nasopharynx) of the mice. Bacteria were
recovered from the nasopharynx by flushing the nostrils with 2 ml of sterile PBS (26), and
lungs were removed from the body and homogenized in 2 ml of sterile PBS using a hand held
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