H O S T M I C R O B E I N T E R A C T I O N

Innate immunity cues virulence Salmonella intestinal pathogens employ a clever trick. They use the immune response that their host triggers to destroy them to enhance the expression of genes that mediate the pathogens virulence.

M I C H E L L E M . C . B U C K N E R & B . B R E T T F I N L A Y

Bacterial pathogens use complex strategies to survive and replicate, causing disease as they do so. But how these strategies, which are usually mediated by molecules called virulence factors, are regu-lated during infection is poorly understood. In a paper published in Cell, Arpaia et al.1 elegantly demonstrate that the Typhimurium serovar of the bacterium Salmonella enterica activates the host immune system and then uses this innate response as a signal to induce its own virulence genes.

Salmonella species are Gram-negative intestinal pathogens that cause disease rang-ing from gastroenteritis to typhoid fever, which can be fatal2. They are transmitted mainly

through contaminated food or water, and colonize their hosts intestine. In typhoid fever, the pathogens can cross the intestinal barrier and spread to the spleen and liver by entering and replicating in phagocytic immune cells such as macrophages2.

The innate immune system is crucial for controlling infectious agents. It recognizes general pathogen-associated molecular pat-terns (PAMPs), and its function is to both kill pathogens and alert the adaptive immune sys-tem3. Integral to innate immunity is a family of proteins called Toll-like receptors (TLRs), which recognize various PAMPs and initiate signalling cascades that act to protect the host3.

Mammals have many TLRs, each of which recognizes certain PAMPs. For example, TLR4, found on the cell surface, recognizes

help to direct molecular-design strategies for making improved conductive polymers for device applications. Vogelsang and colleagues findings are obviously of relevance to industri-ally important processes for assembling con-ductive polymer thin films, but in the longer term, they might also be applicable to the con-trol of morphological order in other polymeric functional materials. We expect that surpris-ing discoveries will be made as single-molecule methods are used to explore other subjects in polymer science.

Yi Fu and Joseph R. Lakowicz are in the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.e-mails: yifu@cfs.umaryland.edu; lakowicz@cfs.umaryland.edu

1. Adachi, T. et al. J. Phys. Chem. C 114, 2089620902 (2010).

2. Barbara, P. F. et al. Acc. Chem. Rev. 38, 602610 (2005).

3. Gesquiere, A. J., Lee, Y. J., Yu, J. & Barbara, P. F. J. Phys. Chem. B 109, 1236612371 (2005).

4. Muls, B. et al. ChemPhysChem 6, 22862294 (2005).5. Vogelsang, J., Brazard, J., Adachi, T., Bolinger, J. C. &

Barbara, P. F. Angew. Chem. Int. Edn 50, 22572261 (2011).

6. Albert, J. N. L. & Epps, T. H. Mater. Today 13, 2433 (2010).

7. Conboy, J. C. et al. J. Phys. Chem. B 102, 45164525 (1998).

8. Dickey, K. C., Anthony, J. E. & Loo, Y. L. Adv. Mater. 18, 17211726 (2006).

Annealing processes are attractive tech-niques for improving the material properties of organic semiconductors (such as conduc-tive polymers) for use in transistors, solar cells and other devices6. SVA induces translocations and folding/unfolding of molecules in poly-mer films at ambient temperatures, eventually leading to the formation of macrostructures featuring equilibrated, long-range molecular order7,8. But so far, our understanding of the morphological transformations the changes in polymer-chain conformations that occur during SVA has been sketchy.

Vogelsang et al.5 are the first to use SMS to study real-time conformational changes in single conductive polymer chains during SVA. They investigated a prototype conductive poly-mer (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene]; MEH-PPV) isolated as single molecules in a host matrix (a non-con-ductive polymer, poly(methyl methacrylate); PMMA). The authors observed clear differ-ences in the translational diffusion of single MEH-PPV chains before, during and after SVA. Before and after annealing, single chains were visible as sharp, stationary fluorescent spots, but during annealing, the spots were blurred and danced around freely (Fig. 1).

From these results, Vogelsang et al. con-cluded that MEH-PPV/PMMA films absorb solvent and swell during SVA, and that the annealing process lowers the glass-transition temperature of the film to below ambient tem-perature, which increases chain mobility. The film therefore exists as a heterogeneous mixture of solid and liquid-like phases during anneal-ing. Crucially, the authors were able to monitor the translational motion of single MEH-PPV chains in the solid and liquid-like phases by simply tracking the movement of the fluores-cence spots. Some of the single-chain diffusion was, however, too rapid to be observed using microscopy, but the authors were able to detect this using a technique known as fluorescence correlation spec troscopy. The authors results also showed that SVA generates different kinds of swelling processes in the host matrix, and causes single MEH-PPV polymer chains to diffuse in several different ways.

In the past few years, various studies13 have provided evidence that MEH-PPV mol-ecules adopt numerous configurations within a PMMA host, ranging from complex dis-ordered structures that are roughly cylindri-cal, to highly ordered ones such as toroids and rods. The three-dimensional shape of MEH-PPV polymer chains is expected to have a profound impact on the electronic properties of the molecules and on their individual fluor-escence quantum yields a measure of how much light the chains produce in response to excitation by incoming photons. Vogelsang and colleagues5 found that MEH-PPV poly-mer chains undergo folding and unfolding events between collapsed and extended conformations during SVA. The transition

between these conformations correlates with fluctuations in the fluorescence intensity of single chains, which occur because the fluo-rescence quantum yield of MEH-PPV is lower in collapsed conformations.

The authors also characterized the conforma-tional order of single MEH-PPV chains using fluorescence excitation polarization spectros-copy, which measures variations in fluor escence in different directions. From these data, Vogel-sang et al. concluded that SVA equilibrates MEH-PPV single chains in the matrix towards lower-energy conformations, which ultimately leads to the production of highly ordered con-formations after annealing. The authors used the same technique to analyse vapour-annealed MEH-PPV/PMMA films that had been pre-pared in different solvents. They observed that, as long as SVA was performed using a good solvent (one in which the film is soluble), then single chains could be reorganized into low-energy, high-order conformations regardless of the solvents in which the film was prepared.

The ability to follow annealing effects at the molecular level will undoubtedly improve our understanding of polymer reorganization pro-cesses, and offers a complementary approach to microscopy methods that monitor the sur-face features and textures of thin films. Insights into how single-chain morphology changes in thin films, block co-polymers, or even in highly ordered self-assembled structures, will

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lipopolysaccharide, a component of the bacterial cell wall3. TLR2, which is also located in the cell membrane, recognizes bacterial surface lipoproteins. And TLR9, located in the membrane of cellular organelles called endosomes, recognizes non-methylated CpG sequences in bacterial DNA.

When a TLR binds its specific PAMP, host-cell adaptor molecules such as MyD88 and TRIF are recruited, and downstream signalling is initiated3. TLR signalling helps to resolve infection not just by activating specific bactericidal mechanisms, but also indirectly by inducing the production of pro-inflammatory proteins such as cytokines. The function of these receptors has generally been investigated in cell lines or in mice lacking MyD88 or TRIF. But this can be problematic, because MyD88 and TRIF also act in non-TLR-associated cellular pathways1,3.

Arpaia et al.1 show that Salmonella activates its virulence genes by using as a signal TLR-induced acidification of the phagosome the intracellular compartment that encloses the engulfed pathogen. The molecules encoded by these genes allow the bacterium to replicate in the normally bactericidal phagosome.

The authors find that mice lacking TLRs 2, 4 and 9 (TLR249 mice) were less suscepti-ble to Salmonella infection than those lacking only TLRs 2 and 4 or TLRs 4 and 9. Moreover,

bone-marrow-derived macrophages (BMMs) from TLR249 mice or MyD88TRIF mice were more resistant to Salmonella infection than the equivalent cells from TLR24 mice. In fact, Arpaia et al. report that TLR249 BMMs can support the replication of several intra cellular pathogens, but not Salmonella. These observations indicate the importance of TLR signalling in Salmonella virulence.

How does Salmonella exploit TLR signal-ling? During an infection, these pathogens normally transform the phagosome into a special vacuole, termed the Salmonella- containing vacuole (SCV), in which they replicate2. In TLR249 BMMs, however, SCV formation is impaired, and the bacteria are detected in the cytoplasm and associate with lysosomes intracellular organelles that have bactericidal activity (Fig. 1).

Salmonella forms, and survives within, the SCV by activating a set of genes that occur in a locus within their genome termed SPI-2. These genes encode a type-III secretion system that functions as a syringe to translocate SPI-2 effector proteins from the bacterial cytoplasm into the host-cell cytoplasm2,4. The effectors manipulate the host cell to allow bacterial rep-lication2. Arpaia et al. studied levels of SPI-2 gene expression and effector translocation and found that unlike the case in normal or TLR24 BMMs SPI-2 was not induced

in TLR249 cells. The authors could restore bacterial replication in TLR249 BMMs using a Salmonella mutant that constitutively expresses SPI-2 genes5.

This paper1 clearly shows that, rather than protecting against Salmonella infection, functional TLR signalling contributes to the pathogens full virulence. Arpaia et al.1 shed light on this apparent paradox by demon-strating that the vacuole of TLR249 and MyD88TRIF BMMs does not acidify as rapidly or to the same extent as the SCV of nor-mal and TLR24 BMMs. It is widely accepted6 that Salmonella uses pH as a signal for SPI-2-gene induction and effector translocation. So it seems that the bacteria take advantage of the essential, and additive, effects of TLRs on vacuole acidification to modulate the expression of virulence genes.

The study also raises several questions. What are the exact pathway(s) linking TLRs and vacuolar acidification? Some SPI-2 effectors also manipulate aspects of the host immune system for instance, the activity of the NF-B transcription factor 7. Such specific effectors might also modulate TLR signalling to induce the expression of virulence genes. Alternatively, more-complex regulatory steps may be involved.

And how do TLRs interact with intracellular Salmonella? Does Salmonella release specific ligands such as nucleic acids into the phago-some to activate TLRs? There is a precedent for this in other species8. So it is possible that Salmonella also uses such ligands to induce a robust immune response.

More broadly, do Salmonella and other bacteria exploit other host immune responses? Aparia and colleagues work underlines the importance of collaboration between workers in immunology and microbiology. Together, these research areas are set to shed light on the intricate interactions between pathogens and their hosts.

Michelle M. C. Buckner and B. Brett Finlay are in the Department of Microbiology and Immunology, and at the Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. e-mail: bfinlay@msl.ubc.ca

1. Arpaia, N. et al. Cell 144, 675688 (2011). 2. Haraga, A., Ohlson, M. B. & Miller, S. I. Nature Rev.

Microbiol. 6, 5366 (2008). 3. Yamamoto, M. & Takeda, K. Gastroenterol. Res. Pract.

2010, 240365 (2010). 4. Shea, J. E., Hensel, M., Gleeson, C. & Holden, D. W.

Proc. Natl Acad. Sci. USA 93, 25932597 (1996). 5. Silphaduang, U., Mascarenhas, M., Karmali, M.

& Coombes, B. K. J. Bacteriol. 189, 36693673 (2007).

6. Yu, X.-J., McGourty, K., Liu, M., Unsworth, K. E. & Holden, D. W. Science 328, 10401043 (2010).

7. Le Negrate, G. et al. J. Immunol. 180, 50455056 (2008).

8. Woodward, J. J., Iavarone, A. T. & Portnoy, D. A. Science 328, 17031705 (2010).

Figure 1 | Salmonella and its effect on macrophages. a,In normal macrophages, Toll-like receptors (TLRs) recognize pathogens such as Salmonella and, together with adaptor molecules including MyD88 and TRIF, initiate downstream signalling pathways to trigger appropriate immune responses. Arpaia etal.1 show that activation of TLRs 2, 4 and 9 acidifies the vacuole containing engulfed Salmonella. This in turn induces the expression of bacterial SPI-2 genes and translocation of the effector molecules into the host-cell cytoplasm. SPI-2 activity transforms the vacuole into a replicative niche, allowing bacterial numbers to increase. b,In macrophages lacking these three TLRs (TLR249), the pH of the Salmonella-containing vacuole does not become acidic as rapidly or to the same extent, and SPI-2 is not induced. This has several consequences: bacterial release into the macrophage cytoplasm; vacuole fusion with lysosomes and so bacterial death; and irregularly shaped Salmonella, which correlates with decreased bacterial survival.

NeutralpH

Signalling

Endosome

NeutralpH

AcidicpH

TLR4MyD88TRIF

SPI-2eectors

Vacuole

TLR2

TLR9

a Normal macrophage b TLR249 macrophage

Irregularly shapedSalmonella

Lysosome

No Salmonella replication

Salmonella

Cellmembrane

Cytoplasm

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