Marcos Pires

ABSTRACT

Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivo technology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions.

IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.

Abstract

Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications

Nature Communications, Published online: 20 April 2017; doi:10.1038/ncomms15015

N-acetyl-muramic acid (NAM) is a core component of the bacterial peptidoglycan (PG) cell wall, and is recognised by the innate immune system. Here the authors engineer Gram-negative and Gram-positive bacteria to incorporate a modified NAM into the backbone of PG, which can be labelled with click chemistry for imaging and tracking.

The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood. We report that AcrAB-TolC, the main multidrug efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria.

Nature advance online publication 19 April 2017. doi:10.1038/nature22067

Authors: Joseph M. Castellano, Kira I. Mosher, Rachelle J. Abbey, Alisha A. McBride, Michelle L. James, Daniela Berdnik, Jadon C. Shen, Bende Zou, Xinmin S. Xie, Martha Tingle, Izumi V. Hinkson, Martin S. Angst & Tony Wyss-Coray

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins....

ABSTRACT

There is much controversy about the metabolic state of cells that are tolerant to antibiotics, known as persister cells. In this opinion piece, we offer an explanation for the discrepancy seen: some laboratories are studying metabolically active and growing cell populations (e.g., as a result of nutrient shifts) and attributing the phenotypes that they discern to persister cells while other labs are studying dormant cells. We argue here that the metabolically active cell population should more accurately be considered tolerant cells, while the dormant cells are the true persister population.

Bacteria within communities can interact to organize their behavior. It remains unclear whether such interactions extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to anti-phase oscillations. This results in time-sharing behavior where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources.

Nature Chemical Biology 13, 551 (2017). doi:10.1038/nchembio.2336

Authors: Kai Papenfort, Justin E Silpe, Kelsey R Schramma, Jian-Ping Cong, Mohammad R Seyedsayamdost & Bonnie L Bassler

Nature advance online publication 05 April 2017. doi:10.1038/nature21686

Authors: Shuo Han, Elizabeth A. Schroeder, Carlos G. Silva-García, Katja Hebestreit, William B. Mair & Anne Brunet

Nature Chemistry. doi:10.1038/nchem.2754

Authors: Jan Stöhr, Haifan Wu, Mimi Nick, Yibing Wu, Manasi Bhate, Carlo Condello, Noah Johnson, Jeffrey Rodgers, Thomas Lemmin, Srabasti Acharya, Julia Becker, Kathleen Robinson, Mark J. S. Kelly, Feng Gai, Gerald Stubbs, Stanley B. Prusiner & William F. DeGrado

The self-propagation of misfolded conformations of tau occurs in neurodegenerative diseases, including Alzheimer's disease. The microtubule-binding region, tau244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Now, it has been shown that a 31-residue peptide from tau's R3 domain forms a cross-β conformation that efficiently seeds aggregation of tau244-372 in cells.

ABSTRACT

Bacterial cells are encased in and stabilized by a netlike peptidoglycan (PGN) cell wall that undergoes turnover during bacterial growth. PGN turnover fragments are frequently salvaged by the cells via a pathway referred to as PGN recycling. Two different routes for the recycling of the cell wall sugar N-acetylmuramic acid (MurNAc) have been recognized in bacteria. In Escherichia coli and related enterobacteria, as well as in most Gram-positive bacteria, MurNAc is recovered via a catabolic route requiring a MurNAc 6-phosphate etherase (MurQ in E. coli) enzyme. However, many Gram-negative bacteria, including Pseudomonas species, lack a MurQ ortholog and use an alternative, anabolic recycling route that bypasses the de novo biosynthesis of uridyldiphosphate (UDP)-MurNAc, the first committed precursor of PGN. Bacteria featuring the latter pathway become intrinsically resistant to the antibiotic fosfomycin, which targets the de novo biosynthesis of UDP-MurNAc. We report here the identification and characterization of a phosphatase enzyme, named MupP, that had been predicted to complete the anabolic recycling pathway of Pseudomonas species but has remained unknown so far. It belongs to the large haloacid dehalogenase family of phosphatases and specifically converts MurNAc 6-phosphate to MurNAc. A mupP mutant of Pseudomonas putida was highly susceptible to fosfomycin, accumulated large amounts of MurNAc 6-phosphate, and showed lower levels of UDP-MurNAc than wild-type cells, altogether consistent with a role for MupP in the anabolic PGN recycling route and as a determinant of intrinsic resistance to fosfomycin.

IMPORTANCE Many Gram-negative bacteria, but not E. coli, make use of a cell wall salvage pathway that contributes to the pool of UDP-MurNAc, the first committed precursor of cell wall synthesis in bacteria. This salvage pathway is of particular interest because it confers intrinsic resistance to the antibiotic fosfomycin, which blocks de novo UDP-MurNAc biosynthesis. Here we identified and characterized a previously missing enzyme within the salvage pathway, the MurNAc 6-phosphate phosphatase MupP of P. putida. MupP, together with the other enzymes of the anabolic recycling pathway, AnmK, AmgK, and MurU, yields UDP-MurNAc, renders bacteria intrinsically resistant to fosfomycin, and thus may serve as a novel drug target for antimicrobial therapy.

Nature Chemistry. doi:10.1038/nchem.2749

Authors: Hiroyuki Koide, Keiichi Yoshimatsu, Yu Hoshino, Shih-Hui Lee, Ai Okajima, Saki Ariizumi, Yudai Narita, Yusuke Yonamine, Adam C. Weisman, Yuri Nishimura, Naoto Oku, Yoshiko Miura & Kenneth J. Shea

Abiotic hydrogel polymer nanoparticles with affinity for a key vascular endothelial growth factor (VEGF165) have now been developed. This high-protein affinity was engineered by carefully controlling the amount and the substitution pattern of sulfated N-acetylglucosamines and the inclusion of a hydrophobic group in the monomer.

Abstract

Nature Chemical Biology 13, 396 (2017). doi:10.1038/nchembio.2302

Authors: Kaitlin Schaefer, Leigh M Matano, Yuan Qiao, Daniel Kahne & Suzanne Walker

Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA. Authors: Roger Tseng, Nicolette F. Goularte, Archana Chavan, Jansen Luu, Susan E. Cohen, Yong-Gang Chang, Joel Heisler, Sheng Li, Alicia K. Michael, Sarvind Tripathi, Susan S. Golden, Andy LiWang, Carrie L. Partch

Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo...

Pentamidine sensitizes Gram-negative pathogens to antibiotics and overcomes acquired colistin resistance

Nature Microbiology, Published online: 6 March 2017; doi:10.1038/nmicrobiol.2017.28

A screen for outer-membrane permeating compounds finds that the clinically approved antiprotozoal drug pentamidine sensitizes drug-resistant Gram-negative pathogens to new antibiotic classes.