Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates

Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection

Abstract: Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-XL, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells. Author Summary: A variety of physiological death signals, as well as pathological insults, trigger apoptosis, a genetically programmed form of cell death. Pathogens often induce host cell apoptosis to establish a successful infection. Neisseria gonorrhoeae (Ngo), the etiological agent of the sexually transmitted disease gonorrhoea, is a highly adapted obligate human-specific pathogen and has been shown to induce apoptosis in infected cells. Here we unveil the molecular mechanisms leading to apoptosis of infected cells. We show that Ngo-mediated apoptosis requires a special subset of proapoptotic proteins from the group of BH3-only proteins. BH3-only proteins act as stress sensors to translate toxic environmental signals to the initiation of apoptosis. In a siRNA-based miniscreen, we found Bim and Bmf, BH3-only proteins associated with the cytoskeleton, necessary to induce host cell apoptosis upon infection. Bim and Bmf inactivated different inhibitors of apoptosis and thereby induced cell death in response to infection. Our data unveil a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic cell death

Long-Range Chromosome Organization in E. coli: A Site-Specific System Isolates the Ter Macrodomain

The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery

Investigating the Responses of Human Epithelial Cells to Predatory Bacteria

One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-alpha levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells.ope

Structural diversity of supercoiled DNA

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA min icircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions

A recent workshop titled “Developing Models to Study Polymicrobial Infections,” sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 351 investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally

Oligomeric Coiled-Coil Adhesin YadA Is a Double-Edged Sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadAV98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions

Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection

Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli

Analysis of In-Vivo LacR-Mediated Gene Repression Based on the Mechanics of DNA Looping

Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58–156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes

Yersinia enterocolitica Serum Resistance Proteins YadA and Ail Bind the Complement Regulator C4b-Binding Protein

Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host