pneumonia

pneumoniajourn

Vol7SpecialIssueforweb

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

and provides insights into the molecular mechanisms of Fn engagement during pneumococcal-host interactions.

P1.17

Pneumolysin-induced nitric oxide contributes to lysosomal membrane

permeabilisation in Streptococcus pneumoniae-infected macrophages

Joseph Ford, Martin Bewley, Simon Johnston, David Dockrell

University of Sheffield, Sheffield, UK

A key phase in the macrophage response to Streptococcus pneumoniae is a program of host-mediated apoptosis. This

occurs after the macrophage has engulfed bacteria, but becomes overwhelmed. This apoptotic response increases

late stage killing of bacteria when conventional phagolysosomal killing has become exhausted. The apical event in

this pathway is lysosomal membrane permeabilisation (LMP). This is caused by a currently unknown mechanism that

requires the pneumococcal toxin pneumolysin (PLY). PLY is a pore-forming toxin but pore-formation is not necessary

to induce LMP, indicating other aspects of PLY activity may be responsible. PLY is known to contribute to nitric oxide

(NO) production in macrophages. We hypothesised that PLY-induced NO production by the macrophage contributes

to LMP and that this is integral to the host response to pneumococcus. To study this system we employed the use of

primary human monocyte derived macrophages (MDMs). MDMs were infected at an MOI of 10 using opsonised D39

S. pneumoniae, a PLY negative mutant (Stop), or latex beads. MDMs were assayed for LMP using flow cytometery and

western blotting. In some experiments MDMs were treated with inhibitors of ROS (Trolox, DPI) and NO (1400W), NO

donors SNAP or NOC-13 and/or TLR agonists lipoteichoic acid (LTA) and lipopolysaccharide (LPS). D39 infected MDM

displayed significantly higher levels of LMP than Stop infected cells. Incubation with 1400W and Trolox significantly

reduced LMP in D39 infected cells, but DPI had no effect. Conversely, incubation with SNAP and NOC-13 was able to

restore LMP in Stop infected cells. Preliminary data suggests that NO is necessary but not sufficient for LMP, with TLR

activation and phagocytosis also required for NO to exert its effect; however, further verification is required. These data

identify a novel role for NO in LMP and host–pneumococcus interactions.

P1.18

Structure of the pneumococcal L,D-carboxypeptidase DacB and impact

of DacB on pathophysiological processes

Mohammed R. Abdullah 1 , Javier Gutiérrez-Fernández 2 , Thomas Pribyl 1 , Nicolas Gisch 3 , Malek

Saleh 1 , Manfred Rohde 4 , Lothar Petruschka 1 , Gerhard Burchhardt 1 , Dominik Schwudke 3 , Juan

Hermoso 2 , Sven Hammerschmidt 1

1

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

Greifswald, Germany; 2 Department of Crystallography and Structural Biology, Institute of Physical-Chemistry “Rocasolano”, CSIC, Madrid, Spain;

3

Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Bioscience, Borstel, Germany; 4 Department of

Molecular Infection Biology, Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany

The peptidoglycan (PGN) is a complex macromolecule and composed of 2 of the alternating sugars residues,

N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which form oligo-(GlcNAc-MurNAc) glycan

strands. These heteropolymers are cross-linked by short peptides to form a complex three-dimensional scaffold

(murein). The PGN forms the essential exoskeleton needed to maintain the shape and osmotic stability of bacteria.

Pneumococcal cell wall hydrolases such as L,D- and D,D-carboxypeptidases DacB and DacA respectively, have

been shown to be important for cell division and shape. Pneumococcal ΔdacA and ΔdacB single and double

mutants were generated and characterised by immunoblot analysis, growth behaviour, and flow cytometry.

The impact of DacB on virulence was tested by phagocytosis assays by applying acute pneumonia mouse

infection model in conjunction with real-time bioimaging. To assess the muropeptide species a PGN analysis

has been carried out. Importantly, the crystal structure of DacB was solved successfully at high resolution.

The L,D-carobxypeptidase DacB is a surface-exposed lipoprotein and its structure is characterised at the atomic level

showing radically different structure, regulation and catalytic machinery than the pneumococcal D,D-carboxypeptidase

DacA. Importantly, the morphological changes observed in dac-mutants are associated with an altered PGN composition

and hence lower bacterial fitness under infection-related conditions. The in vivo mouse infections and cell cultured-based

pneumonia 2015 Volume 7

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