Marcos Pires

Article

Mapping protein-protein interaction using crosslinking and mass spectroscopy strategies is hampered by a high rate of false-positive results. Here, the authors develop a genetically encoded photo-affinity probe for accurate identification of protein interaction partners and crosslinking sites.

Nature Communications doi: 10.1038/ncomms12299

Authors: Yi Yang, Haiping Song, Dan He, Shuai Zhang, Shizhong Dai, Shixian Lin, Rong Meng, Chu Wang, Peng R. Chen

ABSTRACT

Spiroplasma bacteria are highly motile bacteria with no cell wall and a helical morphology. This clade includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. S. poulsonii bacteria are mainly found in the hemolymph of infected female flies and exhibit efficient vertical transmission from mother to offspring. As is the case for many facultative endosymbionts, S. poulsonii can manipulate the reproduction of its host; in particular, S. poulsonii induces male killing in Drosophila melanogaster. Here, we analyze the morphology of S. poulsonii obtained from the hemolymph of infected Drosophila. This endosymbiont was not only found as long helical filaments, as previously described, but was also found in a Y-shaped form. The use of electron microscopy, immunogold staining of the FtsZ protein, and antibiotic treatment unambiguously linked the Y shape of S. poulsonii to cell division. Observation of the Y shape in another Spiroplasma, S. citri, and anecdotic observations from the literature suggest that cell division by longitudinal scission might be prevalent in the Spiroplasma clade. Our study is the first to report the Y-shape mode of cell division in an endosymbiotic bacterium and adds Spiroplasma to the so far limited group of bacteria known to utilize this cell division mode.

IMPORTANCE Most bacteria rely on binary fission, which involves elongation of the bacteria and DNA replication, followed by splitting into two parts. Examples of bacteria with a Y-shape longitudinal scission remain scarce. Here, we report that Spiroplasma poulsonii, an endosymbiotic bacterium living inside the fruit fly Drosophila melanogaster, divide with the longitudinal mode of cell division. Observations of the Y shape in another Spiroplasma, S. citri, suggest that this mode of scission might be prevalent in the Spiroplasma clade. Spiroplasma bacteria are wall-less bacteria with a distinctive helical shape, and these bacteria are always associated with arthropods, notably insects. Our study raises the hypothesis that this mode of cell division by longitudinal scission could be linked to the symbiotic mode of life of these bacteria.

Article

S. Typhimurium can grow selectively on tumours and decreases the cellular levels of the multidrug resistance transporter Pgp. Here, the authors reveal the SipA-dependent mechanism of Pgp down-regulation and produce a SipA-based gold nanoparticle that increases sensitivity to the anticancer drug doxorubicin.

Nature Communications doi: 10.1038/ncomms12225

Authors: Regino Mercado-Lubo, Yuanwei Zhang, Liang Zhao, Kyle Rossi, Xiang Wu, Yekui Zou, Antonio Castillo, Jack Leonard, Rita Bortell, Dale L. Greiner, Leonard D. Shultz, Gang Han, Beth A. McCormick

Article

Exosomes have been identified as promising vehicles for the in vivo delivery of therapeutic molecules. Here the authors design a system to load protein cargos into exosomes during their biogenesis using optogenetic control of protein-protein interactions between the cargo and an exosome-localized partner.

Nature Communications doi: 10.1038/ncomms12277

Authors: Nambin Yim, Seung-Wook Ryu, Kyungsun Choi, Kwang Ryeol Lee, Seunghee Lee, Hojun Choi, Jeongjin Kim, Mohammed R. Shaker, Woong Sun, Ji-Ho Park, Daesoo Kim, Won Do Heo, Chulhee Choi

Cell membranes have a vast repertoire of phospholipid species whose structures can be dynamically modified by enzymatic remodeling of acyl chains and polar head groups. Lipid remodeling plays important roles in membrane biology and dysregulation can lead to disease. Although there have been tremendous advances in creating artificial membranes to...

Nature advance online publication 20 July 2016. doi:10.1038/nature18930

Authors: M. Omar Din, Tal Danino, Arthur Prindle, Matt Skalak, Jangir Selimkhanov, Kaitlin Allen, Ellixis Julio, Eta Atolia, Lev S. Tsimring, Sangeeta N. Bhatia & Jeff Hasty

The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body. It is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbour disease and thus provide a natural platform for the development of engineered therapies. Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ. Here we engineer a clinically relevant bacterium to lyse synchronously at a threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We used microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. As a proof of principle, we tracked the bacterial population dynamics in ectopic syngeneic colorectal tumours in mice via a luminescent reporter. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies, we orally administered the lysis strain alone or in combination with a clinical chemotherapeutic to a syngeneic mouse transplantation model of hepatic colorectal metastases. We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour activity along with a marked survival benefit over either therapy alone. Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites.

Lipoprotein RcsF is the OM component of the Rcs envelope stress response. RcsF exists in complexes with β-barrel proteins (OMPs) allowing it to adopt a transmembrane orientation with a lipidated N-terminal domain on the cell surface and a periplasmic C-terminal domain. Here we report that mutations that remove BamE or alter a residue in the RcsF trans-lumen domain specifically prevent assembly of the interlocked complexes without inactivating either RcsF or the OMP. Using these mutations we demonstrate that these RcsF/OMP complexes are required for sensing OM outer leaflet stress. Using mutations that alter the positively charged surface-exposed domain, we show that RcsF monitors lateral interactions between lipopolysaccharide (LPS) molecules. When these interactions are disrupted by cationic antimicrobial peptides, or by the loss of negatively charged phosphate groups on the LPS molecule, this information is transduced to the RcsF C-terminal signaling domain located in the periplasm to activate the stress response.

DOI: http://dx.doi.org/10.7554/eLife.15276.001

Nature advance online publication 13 July 2016. doi:10.1038/nature18646

Authors: Helle Krogh Pedersen, Valborg Gudmundsdottir, Henrik Bjørn Nielsen, Tuulia Hyotylainen, Trine Nielsen, Benjamin A. H. Jensen, Kristoffer Forslund, Falk Hildebrand, Edi Prifti, Gwen Falony, Emmanuelle Le Chatelier, Florence Levenez, Joel Doré, Ismo Mattila, Damian R. Plichta, Päivi Pöhö, Lars I. Hellgren, Manimozhiyan Arumugam, Shinichi Sunagawa, Sara Vieira-Silva, Torben Jørgensen, Jacob Bak Holm, Kajetan Trošt, MetaHIT Consortium, Karsten Kristiansen, Susanne Brix, Jeroen Raes, Jun Wang, Torben Hansen, Peer Bork, Søren Brunak, Matej Oresic, S. Dusko Ehrlich & Oluf Pedersen

ABSTRACT

Outer membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employing Salmonella enterica serovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in a pagL mutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.

IMPORTANCE The role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis in S. Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. An S. Typhimurium pagL strain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors,...

Antibiotic combinations for controlling colistin-resistant Enterobacter cloacae

The Journal of Antibiotics advance online publication, July 6 2016. doi:10.1038/ja.2016.77

Authors: Thais Bergamin Lima, Osmar Nascimento Silva, Keyla Caroline de Almeida, Suzana Meira Ribeiro, Dielle de Oliveira Motta, Simone Maria-Neto, Michelle Brizolla Lara, Carlos Roberto Souza Filho, Alicia Simalie Ombredane, Celio de Faria Junior, Nadia Skorupa Parachin, Beatriz Simas Magalhães & Octávio Luiz Franco

ABSTRACT

Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against -secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

IMPORTANCE E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.

Article

The role of guanylate-binding proteins (GBPs) in innate immunity is increasingly recognized. Here the authors show that GBP4 activates inflammasome in zebrafish neutrophils, and that this process is critical for the clearance of Salmonella infection via prostaglandin D2.

Nature Communications doi: 10.1038/ncomms12077

Authors: Sylwia D. Tyrkalska, Sergio Candel, Diego Angosto, Victoria Gómez-Abellán, Fátima Martín-Sánchez, Diana García-Moreno, Rubén Zapata-Pérez, Álvaro Sánchez-Ferrer, María P. Sepulcre, Pablo Pelegrín, Victoriano Mulero

Article

Accurate treatment of traumatic brain injuries, a leading cause of neurological disability and death in young people, is hampered by poor accumulation of drugs in the brain. Here, the authors describe a tetrapeptide that can efficiently target brain injuries and deliver therapeutic or diagnostic payload.

Nature Communications doi: 10.1038/ncomms11980

Authors: Aman P. Mann, Pablo Scodeller, Sazid Hussain, Jinmyoung Joo, Ester Kwon, Gary B. Braun, Tarmo Mölder, Zhi-Gang She, Venkata Ramana Kotamraju, Barbara Ranscht, Stan Krajewski, Tambet Teesalu, Sangeeta Bhatia, Michael J. Sailor, Erkki Ruoslahti

The metabolic enzyme hexokinase unexpectedly acts as a pattern recognition receptor that recognizes bacterial peptidoglycan and triggers activation of inflammasomes.

Abstract

In prokaryotes and eukaryotes, cell–cell communication and recognition of self are critical to coordinate multicellular functions. Although kin and kind discrimination are increasingly appreciated to shape naturally occurring microbe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here, we identify a mechanism of interbacterial signal transduction...

Article

Placement of the bacterial division site is crucial for the creation of identical daughter cells. Here, the authors solve the structure of the MapZ protein, which helps to position the cell division protein FtsZ at the cell centre, and further analyse the function of the protein in vivo .

Nature Communications doi: 10.1038/ncomms12071

Authors: Sylvie Manuse, Nicolas L. Jean, Mégane Guinot, Jean-Pierre Lavergne, Cédric Laguri, Catherine M. Bougault, Michael S. VanNieuwenhze, Christophe Grangeasse, Jean-Pierre Simorre

Article

While reversal of lysine methylation on histone tails is a well-established mechanism to tune gene expression, the existence of a similar arginine demethylation process is controversial. Here, the authors show that some jumonji enzymes possess both lysine and arginine demethylase activity in vitro .

Nature Communications doi: 10.1038/ncomms11974

Authors: Louise J. Walport, Richard J. Hopkinson, Rasheduzzaman Chowdhury, Rachel Schiller, Wei Ge, Akane Kawamura, Christopher J. Schofield

Nature Chemistry. doi:10.1038/nchem.2554

Authors: Shigeki Kiyonaka, Ryou Kubota, Yukiko Michibata, Masayoshi Sakakura, Hideo Takahashi, Tomohiro Numata, Ryuji Inoue, Michisuke Yuzaki & Itaru Hamachi

The controlled activation of proteins inside living cells is an important goal in protein design research. Now, a strategy for allosteric activation using coordination chemistry has been demonstrated for two different kinds of neurotransmitter receptors, an ion-channel and a G-protein coupled glutamate receptor.

Metabolic rewiring and directed evolution lead to a fully functional, non-native carbon fixation cycle, which synthesizes sugars and other major biomass components in E. coli.

ABSTRACT

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower K_m at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions.

IMPORTANCE Escherichia coli requires peptidoglycan dd-carboxypeptidases to maintain cell shape by controlling the amount of pentapeptide substrates available to the peptidoglycan synthetic transpeptidases. Why E. coli has eight, seemingly redundant dd-carboxypeptidases has remained unknown. We now show that one of these dd-carboxypeptidases, PBP6b, is important for cell shape maintenance in acidic growth medium, consistent with the higher activity and stability of the enzyme at low pH. Hence, the presence of multiple dd-carboxypeptidases with different enzymatic properties may allow E. coli to maintain a normal cell shape under various growth conditions.

Ogipeptins, novel inhibitors of LPS: physicochemical properties and structural elucidation

The Journal of Antibiotics advance online publication, June 22 2016. doi:10.1038/ja.2016.61

Authors: Yuki Hirota-Takahata, Shiho Kozuma, Nahoki Kuraya, Daisuke Fukuda, Mutsuo Nakajima, Toshio Takatsu & Osamu Ando