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Ubc13 is a ubiquitin-conjugating enzyme that plays a key role in the nuclear factor-κB signal transduction pathway in human diseases. The Shigella flexneri effector OspI affects inflammatory responses by catalyzing the deamidation of a specific glutamine residue at position 100 in Ubc13 during infection. This modification prevents the activation of the TNF (tumor necrosis factor) receptor-associated factor 6, leading to modulation of the diacylglycerol-CBM (CARD-Bcl10-Malt1) complex-TNF receptor-associated factor 6-nuclear factor-κB signaling pathway. To elucidate the structural basis of OspI function, we determined the crystal structures of the catalytically inert OspI C62A mutant and its complex with Ubc13 at resolutions of 3.0 and 2.96Å, respectively. The structure of the OspI-Ubc13 complex revealed that the interacting surfaces between OspI and Ubc13 are a hydrophobic surface and a complementary charged surface. Furthermore, we predict that the complementary charged surface of OspI plays a key role in substrate specificity determination., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The intestinal mucosa is equipped with multiple innate immune defense systems that sense bacterial infection, transmit alarm signals to the immune system, defeat intruding bacteria, and renew damaged and aging epithelial cells. Nevertheless, mucosal bacterial pathogens have versatile pathogenic mechanisms that modulate the host inflammatory and immune responses, manipulate host cell death and survival signal pathways, and renovate the injured epithelium. These properties enable pathogens to adapt to the intestinal mucosal environment, exploit cellular and immune functions, and facilitate infection. Here we review current topics on host defense mechanisms against bacterial infection and the countermeasures that Shigella use to evade the innate immune system., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cycle inhibiting factor (Cif) is one of the effectors delivered into epithelial cells by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) via the type III secretion system (TTSS). Cif family proteins, which inhibit host cell-cycle progression via mechanisms not yet precisely understood, are highly conserved among EPEC, EHEC, Yersinia pseudotuberculosis, Photorhabdus luminescens and Burkholderia pseudomallei. Levels of several proteins relevant to cell-cycle progression are modulated by Cullin-RING ligases (CRLs), which in turn are activated by conjugation and deconjugation of NEDD8 to Cullins. Here we show that Cif interacts with NEDD8 and interferes with SCF (Skp1-Cullin1-F-box protein) complex ubiquitin ligase function. We found that neddylated Cullin family proteins accumulated and ubiquitination of p27 decreased in cells infected with EPEC. Consequently, Cif stabilized SCF substrates such as CyclinD1, Cdt1, and p27, and caused G1 cell-cycle arrest. Using time-lapse-imaging of fluorescent ubiquitination-based cell-cycle indicator (Fucci)-expressing cells, we were able to monitor cell-cycle progression during EPEC infection and confirmed the arrest of infected cells at G1. Our in vitro and in vivo data show that Cif-NEDD8 interaction inhibits deneddylation of Cullins, suppresses CRL activity and induces G1 arrest. We thus conclude that the bacterial effector Cif interferes with neddylation-mediated cell-cycle control., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.

Sequences near the ends of the insertion element IS50 are essential for its transposition, probably because they serve as sites upon which the IS50-encoded transposase protein acts. To determine if these essential sequences include the first base-pair at each end of IS50 we generated 5'C to 5'G transversions at these positions. Each mutation reduced the transposition frequency to 1% to 2% of wild-type. DNA sequence analyses showed that the mutant 5'G is preserved during transposition.

IncFII plasmids consisting of a basic replicon and of an additional fragment(s) unrelated to plasmid maintenance were all less stable in polA1 than in its wild type. Reversion to UV-resistance recovered stability and vice versa. UV irradiation promoted instability in polA1 cells. Larger plasmids showed a greater instability and a fewer number of copies in a same host. Surprisingly, polA1 cells with Tn3 on the plasmid showed a higher ampicillin resistance than the wild type, apparently suggesting that the polA1 mutation increases the copy number. The size-dependency of instability was less marked in polA1 than in its parent. Comparison of the generation times has suggested a detrimental effect exerted by a basic replicon in polA1 hosts.

In a strain of Salmonella typhi isolated from a patient with post-operative typhoid fever, a 217-Kb conjugative plasmid has been detected which codes for lactose fermentation and drug resistance to sulphonamides (SA), chloramphenicol (CP), ampicillin (AP), kanamycin (KM) and trimethoprim (TP). The physical and genetic map was constructed. The lac region containing the lacI gene, the regulatory region and the lacZ and lacY genes was shown to be structurally identical to that of Escherichia coli K12. A transposon carrying genes for resistance to CP and AP, as well as four copies of IS1-like elements, were detected and localized on the plasmid. Their role in the emergence of such a large plasmid is discussed.

In transposition of Tn5 from lambda b221 cI857 Pam Oam rex::Tn5 to the chromosome, lon mutants of Escherichia coli K12 are less efficient than lon+ parents. Reduced frequency of transposition of Tn5 is recovered to the wild type level by adding DL-pantoyl lactone, an agent which suppresses Lon- phenotypes. A suppressor mutation, sulA (or sfiA), suppresses the deficiency of transposition as well as other known lon-associated phenotypes, whereas a lon sulB (or sfiB) double mutant is phenotypically Lon+ but still transposition-deficient. An additive effect of the sulA genes on teh chromosome and on a high copy number plasmid is found in inhibiting transposition of Tn5. A hypothesis that the SulA product someway regulates transposition of Tn5 negatively is proposed.