HIGHLIGHTS BACTERIAL PATHOGENICITY Although structurally similar, the the authors have shown that both LT toxins secreted by enterotoxigenic and CT can bind to the outer mem- Escherichia coli (ETEC) and Vibrio brane of ETEC, but that neither can cholerae — heat-stable enterotoxin bind to the surface of V. cholerae — Vesicles in virulence (LT) and cholera toxin (CT), respec- indicating that the differences in tively — cause diseases of markedly toxin binding are due to differences differing severity in humans. Now, between ETEC and V. cholerae LPS. reporting in the Journal of Biological Furthermore, they found that both Chemistry,researchers at Duke LT and CT bind to the LPS compo- University have shown that struc- nent of the ETEC outer membrane, tural differences in the 3-deoxy- and that the presence of the Kdo LPS D-manno-octulosonic acid (Kdo) core was sufficient for toxin binding. core of the lipopolysaccharide (LPS) Analysis of the structures of component of V. cholerae and ETEC ETEC and V. cholerae LPS showed outer membranes determines the that V. cholerae LPS contains a single extracellular fate of the two toxins, phosphorylated Kdo molecule and postulate that this is responsible bound to lipid A, whereas the LPS of for the differing toxicities. ETEC contains two unphosphory- Both LT and CT are secreted lated Kdo molecules bound to lipid across the outer membrane by the A. By constructing mutant E. coli general secretory pathway; however, and V. cholerae strains, the authors Meta Kuehn and co-workers have showed that LT can bind to an E. coli found that LT remains associated strain with a single Kdo molecule, with the outer membrane of ETEC, but not to an E. coli strain with a whereas CT is released from the phosphorylated Kdo molecule. V. cholerae cell. Now,using an Furthermore, LT was found to bind LT–bacterial-surface-binding assay, to a V. cholerae strain that expressed BACTERIAL PHYSIOLOGY Regulating osmosensing The mechanosensitive (MS) channels MscS not only by increasing osmolarity in the directly. Further confirmation of the and MscL have an important role in external environment, but also by entry importance of RpoS was obtained from bacterial osmoregulation and their into stationary phase. The alternative experiments showing that stationary phase expression is regulated directly by the sigma factor RpoS (also known as σs) is cultures of an rpoS null mutant lysed when stationary phase sigma factor, according to known to be involved in the survival of exposed to hypoosmotic shock conditions. a recent paper in Proc. Natl Acad. Sci. USA. bacterial cells during stationary phase. These new data indicate a strong link In Bacteria, two major families of MS Stokes et al.decided to test whether RpoS between MS channel activity and channels are known — the MscS and MscL could be involved in the induction of the RpoS-dependent cell wall remodelling in families. These gated membrane channels MS channel proteins. Using an rpoS::Tn10 bacterial cells. regulate cell turgor in response to changes mutant, they found that in the absence of In recent years, our understanding of the in the osmolarity of the external RpoS, MscS and MscL expression was molecular mechanisms involved in environment — to relieve excess turgor greatly reduced at high osmolarity and bacterial ‘osmosensing’ has advanced during a decrease in external osmolarity during stationary phase. An increase in the greatly. This new work, which concludes (hypoosmotic shock), MS channels release osmolarity of the culture medium and that the RpoS-mediated regulation of MS solutes from the cell. To avoid cellular entry into stationary phase also increased channel protein expression is required damage, this bacterial ‘osmosensing’ must the levels of β-galactosidase expression during both osmotic stress and entry into occur on a millisecond timescale, and from mscS–lacZ and mscL–lacZ fusion stationary phase, continues the momentum de novo synthesis of the MS channels is constructs. in this field. therefore ruled out. The central role of RpoS in the regulation Sheilagh Clarkson To investigate the regulation of mscS and of MS channel expression was confirmed mscL expression, Stokes et al.used anti- by the fact that two other mutations (clpP References ORIGINAL RESEARCH PAPER Stokes, N. R. et al. A role MscS and anti-MscL antibodies to probe and rssB) that are known to increase the for mechanosensitive channels in survival of stationary membrane preparations from Escherichia stability of RpoS also increased the phase: regulation of channel expression by RpoS. Proc. Natl coli cultured under a variety of growth abundance of MscS and MscL. In vitro Acad. Sci. USA 100, 15959–15964 (2003) FURTHER READING Pivetti, C. D. et al. Two families of conditions. The expression of these MS primer extension revealed that the RpoS mechanosensitive channel proteins. Microbiol. Mol. Biol. channel proteins was found to be induced sigma factor transcribes mscS and mscL Rev. 67, 66–85 (2003) 86 | FEBRUARY 2004 | VOLUME 2 www.nature.com/reviews/micro two Kdo molecules — all of which indicates that toxin binding is inhibited by phosphorylation. The authors propose that this structural difference in the Kdo LPS core of V. cholerae and ETEC is, at least in part, responsible for the dif- fering toxicities of LT and CT — because by being confined to the ETEC outer membrane, delivery of LT in an infected host is restricted to delivery by outer membrane vesi- cles — and propose that transport by vesicles should be considered a specific secretion mechanism for virulence factors. Jane Saunders IMMUNE EVASION References and links ORIGINAL RESEARCH PAPER Horstman, A. et al. Lipopolysaccharide 3-deoxy-D-manno- Proteasome power octulosonic acid (Kdo) core determines bacterial association of secreted toxins. J. Biol. Chem. (2003) doi: 10.1074/jbc.M308633200 Areport in Science has revealed that the An aerosol infection protocol was used to infect FURTHER READING Kaper, J. B., Nataro, J. P. & –/– Mobley, H. L. T. Pathogenic Escherichia coli. proteasome of Mycobacterium tuberculosis could both wild-type and iNOS mice with wild-type Nature Rev. Microbiol. 2, 123–140 (2004) have a role in immune evasion. M. tuberculosis or M. tuberculosis bearing the WEB SITE Meta Kuehn’s laboratory: Worldwide, an estimated 2 billion people are proteasome-associated mutations. Several different http://www.biochem.duke.edu/Kuehn/kuehn.html infected with M. tuberculosis, the causative agent assays showed that the virulence of the mutant of tuberculosis (TB). M. tuberculosis is spread by bacteria was severely attenuated. Eight weeks after aerosols — once inhaled, the pathogen travels to infection, the burden of infection in different the lungs where it is engulfed by alveolar organs was assessed. Between 1 and 4 log10 fewer macrophages. The resulting host immune colony-forming units were recovered from the response includes the production of inducible lungs, liver and spleen of mice infected with the nitric oxide synthase (iNOS) and reactive nitrogen Rv2115c or Rv2097c mutants compared with mice intermediates (RNIs). However, in some infected infected with wild-type bacteria. For Rv2115c, individuals, the host response is sufficient to Darwin et al.went on to show that introducing a contain the infection but doesn’t effect clearance single chromosomal copy of the wild-type allele — some mycobacteria evade the response, remain could reverse this effect. Inhibiting the activity of viable and can be reactivated and cause TB many the mycobacterial proteasome with two different years after the initial infection. chemical inhibitors reproduced the RNI-sensitive Darwin et al.were interested in this population phenotype associated with the Rv2115c and of mycobacteria that cause persistent infection Rv2097c mutations, giving confidence to the and, specifically, how they counter the effects of conclusion that the protein products of Rv2115c RNIs. Their starting point was a transposon and Rv2097c are indeed proteasome related. The mutagenesis strategy — a library of more than authors propose Rv2115c should be renamed mpa, 10,000 transposon mutants was screened for for mycobacterial proteasome, and Rv2097c increased sensitivity to a physiologically relevant renamed paf,for proteasome accessory factor. nitrite. 12 mutants were isolated, each of which From this work, it remains unclear whether the had a unique transposon insertion. Of these, protective effects of the mycobacterial proteasome five were found to have insertions in two genes against RNIs are direct or indirect. Nevertheless, it associated with the mycobacterial proteasome, provides us with a new insight into the function of Rv2115c and Rv2097c. The predicted gene the proteasome within M. tuberculosis,about product of Rv2115c is an AAA family ATPase and which little knowledge has previously been Rv2097c is predicted to encode a proteasome available, and additionally has highlighted a accessory factor. potential new target for anti-TB drugs. In vitro,the growth of all five mutants was Sheilagh Clarkson impaired in macrophages from wild-type mice. However, growth was also impaired in References ORIGINAL RESEARCH PAPER –/– macrophages isolated from iNOS mice, Darwin, K. H. et al. The proteasome of Mycobacterium tuberculosis is indicating that the Rv2115c and Rv2097c required for resistance to nitric oxide. Science 302, 1963–1966 (2003) FURTHER READING Stewart, G. R., Robertson, B. D. & Young, D. B. mutations cause M. tuberculosis to become Tuberculosis: a problem with persistence. Nature Rev. Microbiol. 1, sensitive to iNOS-independent stresses. 97–105 (2003) NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | FEBRUARY 2004 | 87.