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Vol7SpecialIssueforweb

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

pyruvate increase would lead to an increase in the intracellular H 2

O 2

, and in turn, in the amounts of reactive oxygen

species (ROS) resulting from the Fenton reaction (Fe 2+ + H 2

O 2

→ Fe 3+ + OH - + OH . ). ROS damage DNA, lipids and proteins.

With moxifloxacin, we observed similar increases in the production of H 2

O 2

and ROS, which contributed to the lethality

of the drug. In accordance, the lethality of moxifloxacin was attenuated in a strain lacking the spxB gene. These results

support the model of a universal mechanism of action for bactericidal antibiotics, including fluoroquinolones. These

observations are also in agreement with our previous findings that levofloxacin, an inhibitor of topoisomerase IV, triggers

the transcriptional activation of iron transport genes. Both drugs stimulate the Fenton reaction in their mechanism of

action, by causing an increase in the concentration of either iron or H 2

O 2

, respectively.

P2.04

Regulation of PavB by TCS08 in Streptococcus pneumoniae

A Gómez 1, 2 , L. Petruschka 1 , G. Gámez 2, 3 , S. Böhm 1 , V. Kluger 1 , A. Klein 1 , S. Hammerschmidt 1

1

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt University of Greifswald,

D-17487, Greifswald, Germany; 2 Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia,

UdeA, Calle 70 No. 52 - 21, 050010, Medellin, Antioquia, Colombia; 3 Genetics, Regeneration and Cancer (GRC) Research Group, University Research

Center (SIU), Universidad de Antioquia, UdeA, Calle 70 No. 52 - 21, 050010, Medellin, Antioquia, Colombia

The human pathogen Streptococcus pneumoniae (pneumococci) possess 13 two-component regulatory systems (TCSs),

which are crucial for bacterial fitness and virulence. Traditionally, these systems consist of a sensor histidine kinase

(HK) and an output, the response regulator (RR). For TCS08, its encoding genes are placed downstream of the coding

sequence of the adhesin PavB (pneumoccal adherence and virulence factor b), which has been associated with virulence

in the pneumococci [1]. Hence, the interaction of the TCS08 proteins and its effect on the regulation of PavB and other

pneumococcal surface proteins was evaluated in this work. This study aimed to assess the interaction and regulation of

TCS08 proteins and PavB. Maltose binding protein and affinity chromatography were used for TCS08 and PavB proteins

purification. Phosphotransfer profiling and EMSA were established to assess the interaction between the recombinant

HK08 and RR08, and the RR08 and the promoter region of pavB, respectively. The purity of recombinant MBP-HK08

and MBP-RR08 were determined by SDS-PAGE and immunoblot analysis. The HK08 and its cognate RR08 displayed a

phosphorylation interaction, suggesting a phosphatase activity and autophosphorylation, respectively. The interaction

between the pavB promoter fragment and the non-phosphorylated RR08 showed a shift in the electrophoretic mobility

of pavB. The expression of PavB increased dramatically for the hk08-mutant but no significant differences were

found for the rr08- or tcs08-mutants when compared with the wild-type, suggesting a repressor function of the nonphosphorylated

RR08 for the expression of PavB. The results revealed no direct relation of the TCS08 with virulence and

pathogenicity, but it is involved in the expression of PavB in S. pneumoniae.

1. Jensch I, Gámez G, Rothe M, Ebert S, Fulde M, Somplatzki D et al. PavB is a surface-exposed adhesin of

Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections. Mol Microbiol

2010;77:22–43. http://dx.doi.org/10.1111/j.1365-2958.2010.07189.x. PMID:20444103

P2.05

TpxD serves as a global regulator for pneumococcal response to H 2

O 2

,

and is regulated by CodY

Barak Hajaj 1 , Hasan Yesilkaya 2 , Sulman Shafeeq 3 , Xiangyun Zhi 2 , Rachel Benisty 1 , Oscar Kuipers 3 ,

Nurith Porat 1

1

Pediatric Infectious Disease Unit, Soroka University Medical Center, Faculty of Health Sciences, Department of Microbiology and Immunology,

Ben-Gurion University of the Negev, Beer Sheva, Israel; 2 Department of Infection, Immunity & Inflammation, University of Leicester, Leicester,

UK; 3 Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The

Netherlands

Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during

aerobic growth pneumococci produce high levels of H 2

O 2

, which can have adverse effects on cell viability and DNA, and

influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen

pneumonia 2015 Volume 7

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