Host stress hormone norepinephrine stimulates pneumococcal growth, biofilm formation and virulence gene expression

Abstract

Background: Host signals are being shown to have a major impact on the bacterial phenotype. One of them is the endogenously produced catecholamine stress hormones, which are also used therapeutically as inotropes. Recent work form our laboratories have found that stress hormones can markedly increase bacterial growth and virulence. This report reveals that Streptococcus pneumoniae, a commensal that can also be a major cause of community acquired and nosocomial pneumonia, is highly inotrope responsive. Therapeutic levels of the stress hormone norepinephrine increased pneumococcal growth via a mechanism involving provision of iron from serum-transferrin and inotrope uptake, as well as enhancing expression of key genes in central metabolism and virulence. Collectively, our data suggests that Streptococcus pneumoniae recognises host stress as an environmental cue to initiate growth and pathogenic processes.

Results: Effects of a clinically attainable concentration of norepinephrine on S. pneumoniae pathogenicity were explored using in vitro growth and virulence assays, and RT-PCR gene expression profiling of genes involved in metabolism and virulence.We found that norepinephrine was a potent stimulator of growth, via a mechanism involving norepinephrine-delivery of transferrin-iron and internalisation of the inotrope. Stress hormone exposure also markedly increased biofilm formation. Importantly, gene profiling showed that norepinephrine significantly enhanced expression of genes involved in central metabolism and host colonisation. Analysis of the response of the pneumococcal pspA and pspC mutants to the stress hormone showed them to have a central involvement in the catecholamine response mechanism.

Conclusions: Collectively, our evidence suggests that the pneumococcus has mechanisms to recognise and process host stress hormones to augment its virulence properties. The ability to respond to host stress signals may be important for the pneumococcal transition from colonization to invasion mode, which is key to its capacity to cause life-threatening pneumonia, septicaemia and meningitis.

Figure 1

NE stimulates pneumococcal growth and biofilm formation. Panels A-E show the time course of growth of wildtype S. pneumoniae strain D39, TIGR4, and passaged D39 (D39-p) in serum-medium with (red line) and without (black line) the addition of 10 μM NE, epinephrine or dopamine. Panel F is a microscope image showing how NE increased clumping (cell-cell association) of D39 relative to the control, and also increased viability (shown by reduction in cellular lysis debris). Panel G shows the initial attachment of wildtype D39 after 24, 48 and 72 hrs incubation in serum-medium +/-10 μM NE. Attachment was measured as described in Materials and Methods; key: black bar (control); grey bar (NE); P < 0.05 for the collective values of all the NE vs. Control data points. Similar results were also seen with TIGR4 (data not shown). For all experiments, n = 3.

Figure 2

Mechanism of norepinephrine growth induction. Panel A is a western blot of Tf binding to D39 cultures (+) compared with non-Tf supplemented control cultures (-); the influence of growth phase on Tf binding is also shown: log-phase (L), stationary phase (S). Tf represents purified transferrin and is the positive control, indicated by the arrow. Panel B shows that uptake of Tf-complexed Fe (in the form of ⁵⁵Fe) is increased in the presence of NE, P < 0.05; Panel C shows ³H-NE is internalised by D39. The values shown in panels B and C are means of triplicate counts; n = 3.

Figure 3

Role of the PspA and PspC in pneumococcal catecholamine responsiveness. Panels A-C show the time course of growth of pspA, pspC and pspAC mutants in serum-medium in the absence (black line) or presence of 10 μM NE (red line). Panels D-F shows the pspA (pspA^-), pspC (pspC^-) and pspAC (pspAC^-) mutant uptake of Tf-complexed Fe (in the form of ⁵⁵Fe) +/- NE; note that the wildtype D39 uptake of Tf-Fe in the presence of NE is shown in Figure 2B. Values shown in panels D-F are means of triplicate counts; for all experiments, n = 3.

Figure 4

Involvement of PspA and PspC in norepinephrine uptake. Panels A-C show ³H-NE uptake pspA, pspC and pspAC mutants after 24 hrs incubation in serum-medium as described in Methods in the presence (grey) and absence of NE (black); P < 0.05. Values shown are means of triplicate counts; for all experiments, n = 3.