EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72

species, in that it neither has catalase nor the global regulators of peroxide stress resistance such as OxyR and PerR. In

this study we investigated TpxD’s function as a sensor and global regulator in pneumococcal response to H 2

O 2

. Microarray

analysis revealed that TpxD mediates pneumococcal response to H 2

O 2

at the transcriptional level, as mutation of tpxD

abolished H 2

O 2

mediated gene expression. The mechanism underlying TpxD regulatory function was further elucidated

by replacing the catalytic cysteine 58 with alanine. This mutation prevented tpxD up-regulation upon addition of H 2

O 2


and eliminated pneumococcal response to H 2

O 2

, signifying that cysteine 58 is crucial for TpxD signalling activity. We then

aimed to identify the transcription factor governing tpxD expression under H 2

O 2

stress. Bioinformatic analysis discovered

a putative 15-bp consensus CodY binding site in the upstream region of tpxD-coding sequence. The functionality of this

CodY-box was confirmed by mutations and EMSA analysis. Moreover, no up-regulation of TpxD could be detected in

DcodY challenged with H 2

O 2

. These data identify CodY as the transcription factor modulating tpxD expression under H 2

O 2

stress. We propose a model in which TpxD functions as a mediator, transferring H 2

O 2

signal to down-stream effectors,

thereby controlling other targets involved in oxidative stress response.


Co2+-dependent transcriptional regulation in Streptococcus

pneumoniae: opposite effect of Mn2+ and Co2+ on the expression of the

virulence genes psaBCA, pcpA and prtA

Irfan Manzoor 1 , Sulman Shafeeq 1, 2 , Tomas Kloosterman 1 , Oscar Kuipers 1


University of Groningen, Groningen, The Netherlands; 2 Karolinska Institutet, Dtockhlom, Sweden

Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+)-dependent gene regulation in Streptococcus pneumoniae have

already been studied extensively. However, the effect of the important transition metal ion cobalt (Co2+) on gene

expression of S. pneumoniae has not yet been explored. Here, we study the impact of Co2+-stress on the transcriptome

of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+transport

operon (cbi operon), which we show here to be upregulated under Co2+-stress. By means of transcriptional

lacZ-reporter studies, we confirm that the expression of the cbi operon increases with increasing concentrations of

Co2+. Furthermore, we show that Co2+, as has been shown previously for Zn2+, can induce derepression of the genes

of the PsaR regulon, encoding the Mn2+-uptake system (PsaBCA), the choline-binding protein (PcpA) and the cell-wall

associated serine protease (PrtA). The data also corroborate a role for CzcD as a Co2+-efflux pump.


Differential complement sensitivity of Streptococcus pneumoniae and

Streptococcus mitis

Helina E Marshall 1 , Fernanda C Petersen 2 , Jeremy S Brown 1


University College London, London, UK; 2 University of Oslo, Oslo, Norway

Streptococcus pneumoniae and Streptococcus mitis are naso-oropharyngeal commensals that are genetically similar.

However, S. pneumoniae is highly pathogenic and a common cause of pneumonia and septicaemia, whereas S. mitis

rarely causes disease. We hypothesise that differences in sensitivity to innate immunity may underlie these differences

in virulence phenotype. We compared sensitivity of S. pneumoniae and S. mitis to neutrophil killing. After opsonisation

with serum, but not with heat-treated serum or PBS, S. mitis was markedly more sensitive to neutrophil killing compared

to S. pneumoniae. These differences suggested S. mitis was relatively complement sensitive, and flow cytometry assays of

C3b/iC3b deposition confirmed there was increased complement opsonisation of S. mitis compared to S. pneumoniae. S.

pneumoniae resistance to complement is partially dependent on binding of the immune regulator factor H by the surface

protein PspC. We investigated factor H binding to S. mitis using flow cytometry. The results demonstrated that there

was no significant factor H binding to S. mitis. By inserting pspC of S. pneumoniae into S. mitis, we demonstrated that

expression of PspC enabled S. mitis to then bind factor H. Investigation of C3b/iC3b confirmed a decrease in opsonisation.

Furthermore, survival in whole human blood of this modified strain showed an increase, when compared to the wildtype

strain. These results suggest that an inability to bind factor H might underpin S. mitis sensitivity to opsonisation with

complement and neutrophil killing compared to S. pneumoniae, and therefore contribute to the differences in virulence

between these 2 commensal species.

pneumonia 2015 Volume 7


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