Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane. Functional analysis of IpaC.

Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.

A comparative vibrational and NMR study of cis-cinnamic acid polymorphs and trans-cinnamic acid.

The IR and Raman spectra of the two polymorphic forms (58 degree- and 68 degree-forms) of cis-cinnamic acid were measured, and the spectral differences discussed on the basis of the crystal structures of the two forms. The IR bands related to the COOH group differ in the frequencies and band shape, reflecting differences in the hydrogen bonding between the two modifications. These spectra were compared with those of trans-cinnamic acid. The IR, Raman, and NMR spectra of the isotopic compounds, including the deuterated and 13C analogs of the cis and trans acids, were also recorded in the solid state and in solution to confirm the spectral assignments.

Molecular structure and vibrational spectra of phenolphthalein and its dianion.

Infrared (IR) and Raman spectra of phenolphthalein (PP) and its dianion form (sodium and potassium salts) were studied both in the solid state and in aqueous solution. Band assignments were carried out on the basis of the isotope shifts of the ring deuterated and 13C-substituted derivatives. Spectral analyses reveal that the PP dianion exists as mixtures of the benzenoid form (colorless) and the quinonoid form (colored) in the solid state and in aqueous solution, while the neutral PP solely takes the gamma-lactone form. This work provides the first vibrational spectroscopic evidence for the coexistence of the two species in the PP dianions.

Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors.

We investigated the supramolecular structure of the SHIGELLA: type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F(1)-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937.

Shigella flexneri, but not a non-invasive mutant derivative rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-gamma (IFN gamma) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFN gamma-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RA-differentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFN gamma-pretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state.

The modified nucleoside 2-methylthio-N6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes.

The virulence of the human pathogen Shigella flexneri is dependent on both chromosome- and large-virulence-plasmid-encoded genes. A kanamycin resistance cassette mutation in the miaA gene (miaA::Km Sma), which encodes the tRNA N6-isopentyladenosine (i6A37) synthetase and is involved in the first step of the synthesis of the modified nucleoside 2-methylthio-N6-isopentenyladenosine (ms2i6A), was transferred to the chromosome of S. flexneri 2a by phage P1 transduction. In the wild-type bacterium, ms2i6A37 is present in position 37 (next to and 3' of the anticodon) in a subset of tRNA species-reading codons starting with U (except tRNA(Ser) species SerI and SerV). The miaA::Km Sma mutant of S. flexneri accordingly lacked ms2i6A37 in its tRNA. In addition, the mutant strains showed reduced expression of the virulence-related genes ipaB, ipaC, ipaD, virG, and virF, accounting for sixfold-reduced contact hemolytic activity and a delayed response in the focus plaque assay. A cloned sequence resulting from PCR amplification of the wild-type Shigella chromosome and exhibiting 99% homology with the nucleotide sequence of the Escherichia coli miaA gene complemented the virulence-associated phenotypes as well as the level of the modified nucleoside ms2i6A in the tRNA of the miaA mutants. In the miaA mutant, the level of the virulence-associated protein VirF was reduced 10-fold compared with the wild type. However, the levels of virF mRNA were identical in the mutant and in the wild type. These findings suggest that a posttranscriptional mechanism influenced by the presence of the modified nucleoside ms2i6A in the tRNA is involved in the expression of the virF gene. The role of the miaA gene in the virulence of other Shigella species and in enteroinvasive E. coli was further generalized.