Fig 1.
Temperature influences serum killing and opsonophagocytosis of H. influenzae and S. pneumoniae, respectively.
(A) Serum killing of H. influenzae type b was measured by pre-incubating the bacteria at 30°C or 37°C in RMPI for 1 hour prior to addition of 25% pooled human serum. The mixtures were subjected to serum killing at 37°C for 30 minutes. H. influenzae type b pre-incubated at 37°C shows higher resistance to complement-mediated killing than bacteria at 30°C. (B) The effect of temperature on opsonophagocytosis of pneumococci by THP-1 macrophages was analysed by growing S. pneumoniae TIGR4 bacteria at either 30°C or 37°C. The bacteria were then opsonised with pooled human serum for 30 minutes at 37°C. Non-opsonised bacteria were exposed to only RPMI for 30min at 37°C. Pneumococci grown at 37°C are more resistant to phagocytosis by macrophages than bacteria grown at 30°C. Error bars denote s.e.m. Statistical significance calculated using a paired, two-tailed student t-test.
Fig 2.
Capsular gene expression and production of capsules of S. pneumoniae and H. influenzae are temperature regulated.
(A) Capsular dot blot analyses of S. pneumoniae and H. influenzae type b (Hib) show increased presence of capsular components in culture supernatants with increasing temperature. Pneumococcal Serotype 4 and H. influenzae type b polysaccharide capsule specific anti-sera were used for detection of capsules. (B) The S. pneumoniae capsule is thicker at higher temperature as shown by dextran exclusion assay (right panel, bar = 5μm). The average thickness of pneumococcal capsules (μm) grown at different temperature were tabulated and shown in the graph with standard deviations (+/-). (C) Thermoregulation of CpsA and Bcs1´ UTR-gfp fusion products detected in E. coli by Western blot analysis (Anti-GFP antibody used for detection of GFP, RecA and Neomycin-phosphotransferase used as controls). (D) Western blots of in vitro transcription/translation assay of CpsA and Bcs1´ UTR-gfp fusion products show temperature regulation of CpsA-GFP and Bcs1´-GFP. (Anti-GFP antibody used for detection of GFP). Error bars denote s.e.m. Statistical significance calculated using a paired, two-tailed student t-test.
Fig 3.
Factor H (FH) binding protein expression of S. pneumoniae and H. influenzae is temperature regulated.
(A) Western and far-western blot analyses show thermoregulated PspC and FH expression in S. pneumoniae. Anti-PspC (S. pneumoniae) and anti-PH (H. influenzae) antibodies used for detection of respective FH binding protein. Human FH was used to determine binding to respective FH binding protein. Anti-human FH antibody was used. LytA (S. pneumoniae) and RecA (H. influenzae) were used as loading controls. (B) Western blots of in vitro transcription/translation assay of PspC and PH UTR-gfp fusion products show temperature regulation of PspC-GFP and PH-GFP. (Anti-GFP antibody used for detection of GFP). (C) Fluorescent flow cytometry shows increased human FH binding to S. pneumoniae grown at higher temperature. Fluorescent microscopy analysis reveals that more FH is bound to S. pneumoniae at 40°C (FH binding in a banded pattern). (D) Fluorescent flow cytometry shows increased human FH binding to H. influenzae type b (Hib) grown at higher temperature. Fluorescent microscopy analysis reveals a FH positive population of H. influenzae type b along with a FH negative population at 40°C. (C & D) Fluorescence intensities of three experiments were pooled and FH binding of bacteria grown at 40°C vs 32°C analysed. Error bars denote s.e.m. Statistical significance calculated using a paired, two-tailed student t-test.
Fig 4.
Clinical isolates of S. pneumoniae also possess thermosensing capability.
(A) Thermoregulation of CpsB in S. pneumoniae TIGR4 as detected by western blot analysis. (B) Thermoregulation of CpsB and PspC, using western blot analyses, in clinical pneumococcal isolates of serotypes 1, 2 19F and 22F. LytA was used as a loading control for all panels.
Table 1.
5′-UTR sequences of cpsA, pspC, bcs1´, lph were analysed using nBLAST for occurrences in other isolates. Number of isolates identified (n).
Fig 5.
Model of pathogenesis, from commensalism to systemic infection.
Mucosal surface inflammation (e.g. by trauma or infection) raises the temperature, leading to increased expression of virulence determinants such as polysaccharide capsules and FH binding proteins of S. pneumoniae, H. influenzae and N. meningitidis. This thermoregulation enables the bacteria to evade immune responses on the mucosal surfaces. The bacteria are primed to higher temperature and might have a better chance of surviving within the body, possibly increasing the rate of systemic infections. Green box in RNA structure denotes ribosome binding site. Model not according to scale.
Table 2.
Oligonucleotides, strains and plasmids.