442 | JUNE 2004 | VOLUME 2 www.nature.com/reviews/micro

H I G H L I G H T S

For many bacterial pathogens, adhe-sion to host cells and subsequentinternalization are the two initialsteps in the colonization process. ForStreptococcus pyogenes, binding tofibronection via the cell-wall-attached fibronectin-binding recep-tor, protein F1, is a key molecularinteraction that mediates these steps.Now, a report published in the Mayissue of Microbiology sheds new lighton the regulation of this crucialinteraction by revealing a role for thesecreted bacterial protease, SpeB.

SpeB, which is secreted in largeamounts by most S. pyogenes strains,has broad proteolytic activity againsta number of different human pro-teins, as well as several S. pyogenesproteins that are located on the bac-terial cell surface. Previous studieshave also shown that SpeB con-tributes to bacterial internalization,but the mechanisms by which theprotease influences this process wereunclear. Patrik Nyberg and col-leagues set out to clarify the role of

SpeB in streptococcal entry into hostcells, focusing on the requirement offibronectin. Initially, the authorswere able to demonstrate that grow-ing S. pyogenes in a medium thatpromoted SpeB expression reducedthe ability of the bacteria to bind tofibronectin. They were further ableto show that this reduction infibronectin-binding activity was dueto the proteolysis of protein F1 bySpeB. As bacterial internalization ismediated by protein F1, the authorswere also able to demonstrate thatthe removal of protein F1 from thebacterial cell surface by SpeB resultedin a significant decrease in adhesionand internalization. Unlike othersurface proteins of S. pyogenes thatare protected from proteolytic degra-dation by interacting with their lig-ands, protein F1 was readily cleavedby SpeB, even when complexed withfibronectin.

These data unambiguously showthe sensitivity of the fibronectin-binding activity of S. pyogenes to the

Legionella pneumophila is an intracellularpathogen that can avoid delivery tolysosomes — and certain death — afteruptake by a host cell, by modifying thevacuole that it is constrained within toprovide a safe place for bacterialreplication. Research published in theJournal of Experimental Medicine revealsthat L. pneumophila recruits the hosttrafficking proteins Rab1 and Sec22b to aidin the maturation of the vacuole into areplicative organelle.

After entering the host cell (usually amacrophage) L. pneumophila is confined ina Legionella-containing vacuole (LCV),which later matures into a replicativeorganelle. LCV maturation depends on theexpression of the Dot/Icm bacterialsecretion system, which injects bacterialproteins into the host cell cytosol.Microscopy and bacterial genetics haverevealed that vesicles that exit theendoplasmic reticulum (ER), and aredestined for transport to the Golgi, areredirected to fuse with the LCV. Subsequentremodelling of the LCV results in a

protected niche — the replicative organelle— in which the bacteria multiply.Mechanisms that mediate the LCVmaturation process, including thetransport pathways that the bacteriumaffects, were investigated in this study.

Rab proteins function to transportvesicles between the ER and the Golgi, soKagan et al. used immunofluorescencemicroscopy to investigate whether Rabproteins were recruited to the LCV. Of thethree Rab proteins (Rab1, 2 and 6) thatfunction in vesicular transport between theER and the Golgi only Rab1 was localizedto the LCV. This process depended onfunctioning of the Dot/Icm secretionsystem, but not on the exit of earlysecretory vesicles from the ER, so bacterialfactors injected into the host cellpresumably recruit Rab1. Since treatmentof host cells to prevent vesicular transportdid not prevent Rab1 recruitment to theLCV, Rab1 recruitment is independent ofER vesicle-mediated LCV-remodelling.Abrogation of cellular Rab1 functionreduced the intracellular replication of

L. pneumophila so Rab1 clearly has a role inconversion of the LCV into a competentreplication organelle.

Sec22b, a SNARE protein thatparticipates in vesicular fusion and isrecruited by Rab proteins, was alsorecruited to the LCV, but again only if theDot/Icm system was functional. In contrastwith Rab1 recruitment, Sec22b recruitmentrequired early ER vesicular transport, so itseems that Sec22b is recruited after Rab1.Kagan et al. disrupted Rab1 function, andused immunogold microscopy to visualizeSec22b, to show that Rab1 recruitmentpromotes the transport to, and fusion of,ER-derived vesicles that contain Sec22bwith the LCV.

Dissecting the LCV-maturation processfurther to delineate the recruitment andvesicular fusion mechanisms will shed lightnot only on Legionella pathogenesis, but onthe basic cell biology of phagosomematuration. This study clearly demon-strates the utility of bacterial modelsystems for cell biology research.

Susan JonesReferences and links

ORIGINAL RESEARCH PAPER Kagan, J. C. et al.Legionella subvert the functions of Rab1 and Sec22b tocreate a replicative organelle. J. Exp. Med. 199, 1201–1211(2004)WEB SITECraig Roy’s laboratory:http://info.med.yale.edu/micropath/fac_roy.html

Colonization control

B A C T E R I A L PAT H O G E N E S I S

Adhesion and internalization of Streptococcus pyogenes on an epithelial cell layer asvisualized by scanning electron microscopy. The scale bar represents 2 µm. Bacteria beinginternalized are shown at higher magnification in the insert (scale bar 1µm). Photo courtesyof Matthias Mörgelin, Lund University, Sweden.

Recruitment drive

C E L L U L A R M I C R O B I O LO G Y

H I G H L I G H T S

NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | JUNE 2004 | 443

A new report in Science has revealed another weaponthat viruses can use to control host gene expression:microRNAs.

MicroRNAs (miRNAs) are endogenouslyexpressed, small (~22-nucleotide) RNAs that areknown to regulate gene expression in plants and ani-mals by targeting specific mRNAs for degradation ortranslational repression. Although the first miRNAwas identified in Caenorhabditis elegans in 1993, theubiquitous nature of miRNAs was not discovereduntil 2000. Since then, rapid progress has been made— miRNAs have been identified in all animal andplant genomes analysed to date and miRNAs are now known to be one of the most abundant generegulatory molecules in animals.

Pfeffer et al. looked for evidence of the presence ofmiRNAs in Epstein–Barr virus (EBV), a γ-herpesvirusthat is ubiquitous in human populations. EBVinfects a variety of cell types, including epithelialcells and B cells, where it establishes a latent infec-tion. EBV infection is associated with ~1% of all can-cers worldwide, including Burkitt’s lymphoma (BL)and nasopharyngeal carcinoma.

Pfeffer et al. cloned all the small RNAs from a BLcell line. Sequence analysis of the cloned EBV RNAindicated that the structural features were typical ofthose of miRNAs. Five EBV miRNAs were identifiedin total and are located in two clusters in the EBVgenome. Three are located within the BHFR1 codingregion and have been named miR-BHRF1-1, -2 and -3, and the other two are located in the intronicregion of the BART gene and have been named miR-BART1 and -2.

Latent infection with EBV occurs in distinctstages, during which different viral latent genes are

expressed. To establish whether the EBV miRNAswere expressed during a particular stage, Pfeffer et al.screened a variety of cell lines at different stages oflatent infection. miR-BART1 and -2 were detected atall stages of latency, consistent with the fact the BARTis expressed throughout latent infection. By contrast,the expression of the miR-BHRF1 miRNAs wasfound to be stage-dependent. Interestingly, miR-BHRF1 was detected in one of two cell lines in latentstage I, which were previously thought to expressonly one EBV latent protein and EBERs (small EBV-encoded RNAs); Pfeffer et al. suggest that a newlatent stage might need to be created to distinguishbetween cells that express BHFR1 miRNAs and thosethat do not.

In silico analysis to identify putative host celltargets for these miRNAs identified many possibletargets, including regulators of cell proliferationand apoptosis. Of the putative targets identified,more than half are also targeted by miRNAsencoded by the host cell. Experimental evidencewas also obtained to indicate that at least one of theEBV miRNAs can regulate EBV gene expression.

As viruses have perfected their control over hostcells during millions of years of co-evolution, it isperhaps unsurprising that yet another method thatviruses can use to manipulate host gene expressionhas been uncovered. Using non-coding RNAs toregulate gene expression is not only convenient butalso non-immunogenic and could therefore alsocontribute to viral immune evasion. Pfeffer et al.anticipate that miRNAs will be identified in otherγ-herpesviruses and other viruses with large DNAgenomes.

Sheilagh Clarkson

References and linksORIGINAL RESEARCH PAPER Pfeffer, S. et al. Identification of virus-encoded microRNAs. Science 304, 734–736 (2004)FURTHER READING Bartel, D. P. MicroRNAs: genomics, biogenesis,mechanism and function. Cell 116, 281–297 (2004)

proteolytic action of SpeB with theresulting effects on bacterial internal-ization. This function for SpeB couldrepresent a mechanism for the pro-teolytic regulation of S. pyogenesentry into host cells. Indeed, asSpeB expression and secretion arethought to be induced when thebacteria are in an environment inwhich nutrients are limited, theauthors speculate that proteolysisof protein F1 and the subsequentinhibition of adhesion and inter-nalization, could promote bacterialdissemination by allowing themovement of S. pyogenes to newand more fertile locations.

David O’Connell

References and linksORIGINAL RESEARCH PAPER Nyberg, P. et al.SpeB modulates fibronectin-dependentinternalization of Streptococcus pyogenes byefficient proteolysis of cell-wall-anchored proteinF1. Microbiology 150, 1559–1569 (2004)WEB SITEFree full-text access to this research paper:http://mic.sgmjournals.org/cgi/content/full/150/5/1559Brought to you in collaboration with theSociety for General Microbiology

Micro modulators

V I R A L PAT H O G E N E S I S