Publications by authors named "Howard A Shuman"

50 Publications

GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii.

Infect Immun 2020 12 15;89(1). Epub 2020 Dec 15.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA.

A critical facet of mammalian innate immunity involves the hosts' attempts to sequester and/or limit the availability of key metabolic products from pathogens. For example, nutritional immunity encompasses host approaches to limit the availability of key heavy metal ions such as zinc and iron. Previously, we identified several hundred genes in a multidrug-resistant isolate of that are required for growth and/or survival in the infection model. In the present study, we further characterize one of these genes, a LysR family transcription regulator that we previously named GigC. We show that mutant strains lacking have impaired growth in the absence of the amino acid cysteine and that regulates the expression of several genes involved in the sulfur assimilation and cysteine biosynthetic pathways. We further show that cells harboring a deletion of the gene are attenuated in two murine infection models, suggesting that the GigC protein, likely through its regulation of the cysteine biosynthetic pathway, plays a key role in the virulence of .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.00180-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927930PMC
December 2020

Acinetobacter baumannii NCIMB8209: a Rare Environmental Strain Displaying Extensive Insertion Sequence-Mediated Genome Remodeling Resulting in the Loss of Exposed Cell Structures and Defensive Mechanisms.

mSphere 2020 07 29;5(4). Epub 2020 Jul 29.

Instituto de Biologia Molecular y Celular de Rosario (IBR, CONICET), Departamento de Microbiologia, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Rosario, Argentina

represents nowadays an important nosocomial pathogen of poorly defined reservoirs outside the clinical setting. Here, we conducted whole-genome sequencing analysis of the sp. NCIMB8209 collection strain, isolated in 1943 from the aerobic degradation (retting) of desert guayule shrubs. Strain NCIMB8209 contained a 3.75-Mb chromosome and a plasmid of 134 kb. Phylogenetic analysis based on core genes indicated NCIMB8209 affiliation to , a result supported by the identification of a chromosomal -like gene. Seven genomic islands lacking antimicrobial resistance determinants, 5 regions encompassing phage-related genes, and notably, 93 insertion sequences (IS) were found in this genome. NCIMB8209 harbors most genes linked to persistence and virulence described in contemporary clinical strains, but many of the genes encoding components of surface structures are interrupted by IS. Moreover, defense genetic islands against biological aggressors such as type 6 secretion systems or CRISPR-cas are absent from this genome. These findings correlate with a low capacity of NCIMB8209 to form biofilm and pellicle, low motility on semisolid medium, and low virulence toward and Searching for catabolic genes and concomitant metabolic assays revealed the ability of NCIMB8209 to grow on a wide range of substances produced by plants, including aromatic acids and defense compounds against external aggressors. All the above features strongly suggest that NCIMB8209 has evolved specific adaptive features to a particular environmental niche. Moreover, they also revealed that the remarkable genetic plasticity identified in contemporary clinical strains represents an intrinsic characteristic of the species. is an ESKAPE (, , , , , and species) opportunistic pathogen, with poorly defined natural habitats/reservoirs outside the clinical setting. arose from the - complex as the result of a population bottleneck, followed by a recent population expansion from a few clinically relevant clones endowed with an arsenal of resistance and virulence genes. Still, the identification of virulence traits and the evolutionary paths leading to a pathogenic lifestyle has remained elusive, and thus, the study of nonclinical ("environmental") isolates is necessary. We conducted here comparative genomic and virulence studies on NCMBI8209 isolated in 1943 from the microbiota responsible for the decomposition of guayule, and therefore well differentiated both temporally and epidemiologically from the multidrug-resistant strains that are predominant nowadays. Our work provides insights on the adaptive strategies used by to escape from host defenses and may help the adoption of measures aimed to limit its further dissemination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSphere.00404-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392541PMC
July 2020

Metabolic regulation of gene expression by histone lactylation.

Nature 2019 10 23;574(7779):575-580. Epub 2019 Oct 23.

Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA.

The Warburg effect, which originally described increased production of lactate in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, polarization of macrophages and activation of T cells. This phenomenon is intimately linked to several diseases including neoplasia, sepsis and autoimmune diseases. Lactate, which is converted from pyruvate in tumour cells, is widely known as an energy source and metabolic by-product. However, its non-metabolic functions in physiology and disease remain unknown. Here we show that lactate-derived lactylation of histone lysine residues serves as an epigenetic modification that directly stimulates gene transcription from chromatin. We identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce the production of lactate by glycolysis, and this acts as a precursor that stimulates histone lactylation. Using M1 macrophages that have been exposed to bacteria as a model system, we show that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, increased histone lactylation induces homeostatic genes that are involved in wound healing, including Arg1. Collectively, our results suggest that an endogenous 'lactate clock' in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Histone lactylation thus represents an opportunity to improve our understanding of the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1678-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818755PMC
October 2019

Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections.

Sci Transl Med 2018 09;10(460)

Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195, USA.

The lack of new antibiotics is among the most critical challenges facing medicine. The problem is particularly acute for Gram-negative bacteria. An unconventional antibiotic strategy is to target bacterial nutrition and metabolism. The metal gallium can disrupt bacterial iron metabolism because it substitutes for iron when taken up by bacteria. We investigated the antibiotic activity of gallium ex vivo, in a mouse model of airway infection, and in a phase 1 clinical trial in individuals with cystic fibrosis (CF) and chronic airway infections. Our results show that micromolar concentrations of gallium inhibited growth in sputum samples from patients with CF. Ex vivo experiments indicated that gallium inhibited key iron-dependent bacterial enzymes and increased bacterial sensitivity to oxidants. Furthermore, gallium resistance developed slowly, its activity was synergistic with certain antibiotics, and gallium did not diminish the antibacterial activity of host macrophages. Systemic gallium treatment showed antibiotic activity in murine lung infections. In addition, systemic gallium treatment improved lung function in people with CF and chronic lung infection in a preliminary phase 1 clinical trial. These findings raise the possibility that human infections could be treated by targeting iron metabolism or other nutritional vulnerabilities of bacterial pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aat7520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637966PMC
September 2018

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii.

J Bacteriol 2017 05 25;199(10). Epub 2017 Apr 25.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA.

A critical component of bacterial pathogenesis is the ability of an invading organism to sense and adapt to the harsh environment imposed by the host's immune system. This is especially important for opportunistic pathogens, such as , a nutritionally versatile environmental organism that has recently gained attention as a life-threatening human pathogen. The emergence of is closely linked to antibiotic resistance, and many contemporary isolates are multidrug resistant (MDR). Unlike many other MDR pathogens, the molecular mechanisms underlying pathogenesis remain largely unknown. We report here the characterization of two recently identified virulence determinants, GigA and GigB, which comprise a signal transduction pathway required for surviving environmental stresses, causing infection and antibiotic resistance. Through transcriptome analysis, we show that GigA and GigB coordinately regulate the expression of many genes and are required for generating an appropriate transcriptional response during antibiotic exposure. Genetic and biochemical data demonstrate a direct link between GigA and GigB and the nitrogen phosphotransferase system (PTS), establishing a novel connection between a novel stress response module and a well-conserved metabolic-sensing pathway. Based on the results presented here, we propose that GigA and GigB are master regulators of a global stress response in , and coupling this pathway with the PTS allows to integrate cellular metabolic status with external environmental cues. Opportunistic pathogens, including , encounter many harsh environments during the infection cycle, including antibiotic exposure and the hostile environment within a host. While the development of antibiotic resistance in has been well studied, how this organism senses and responds to environmental cues remain largely unknown. Herein, we investigate two previously identified virulence determinants, GigA and GigB, and report that they are required for stress resistance, likely comprising upstream elements of a global stress response pathway. Additional experiments identify a connection between GigA/GigB and a widely conserved metabolic-sensing pathway, the nitrogen phosphotransferase system. We propose that coordination of these two pathways allows to respond appropriately to changing environmental conditions, including those encountered during infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00066-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405210PMC
May 2017

Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria.

PLoS One 2017 3;12(3):e0173116. Epub 2017 Mar 3.

Department of Microbiology, University of Chicago, Chicago, Illinois, United States of America.

The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires' Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173116PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336243PMC
August 2017

A dual-targeting approach to inhibit Brucella abortus replication in human cells.

Sci Rep 2016 10 21;6:35835. Epub 2016 Oct 21.

Howard Taylor Ricketts Laboratory, University of Chicago, Argonne National Laboratory, Lemont, Illinois, United States of America.

Brucella abortus is an intracellular bacterial pathogen and an etiological agent of the zoonotic disease known as brucellosis. Brucellosis can be challenging to treat with conventional antibiotic therapies and, in some cases, may develop into a debilitating and life-threatening chronic illness. We used multiple independent assays of in vitro metabolism and intracellular replication to screen a library of 480 known bioactive compounds for novel B. abortus anti-infectives. Eighteen non-cytotoxic compounds specifically inhibited B. abortus replication in the intracellular niche, which suggests these molecules function by targeting host cell processes. Twenty-six compounds inhibited B. abortus metabolism in axenic culture, thirteen of which are non-cytotoxic to human host cells and attenuate B. abortus replication in the intracellular niche. The most potent non-cytotoxic inhibitors of intracellular replication reduce B. abortus metabolism in axenic culture and perturb features of mammalian cellular biology including mitochondrial function and receptor tyrosine kinase signaling. The efficacy of these molecules as inhibitors of B. abortus replication in the intracellular niche suggests "dual-target" compounds that coordinately perturb host and pathogen are promising candidates for development of improved therapeutics for intracellular infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep35835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073326PMC
October 2016

Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution of Legionella effectors.

Proc Natl Acad Sci U S A 2016 08 19;113(31):8807-12. Epub 2016 Jul 19.

Department of Microbiology, University of Chicago, Chicago, IL 60637;

Legionella pneumophila, the Gram-negative pathogen causing Legionnaires' disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host's disassembly machinery, thus effectively trapping VAMP4 in an inactive state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1608755113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978295PMC
August 2016

Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires.

Nat Genet 2016 Feb 11;48(2):167-75. Epub 2016 Jan 11.

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

Infection by the human pathogen Legionella pneumophila relies on the translocation of ∼ 300 virulence proteins, termed effectors, which manipulate host cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires using a previously validated machine learning approach. This analysis identified 5,885 predicted effectors. The effector repertoires of different Legionella species were found to be largely non-overlapping, and only seven core effectors were shared by all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from the natural protozoan hosts of these species. Furthermore, we detected numerous new conserved effector domains and discovered new domain combinations, which allowed the inference of as yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050043PMC
http://dx.doi.org/10.1038/ng.3481DOI Listing
February 2016

Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii.

mBio 2015 Nov 10;6(6):e01660-15. Epub 2015 Nov 10.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA.

Unlabelled: The increasing emergence of antibiotic-resistant bacterial pathogens represents a serious risk to human health and the entire health care system. Many currently circulating strains of Acinetobacter baumannii exhibit resistance to multiple antibiotics. A key limitation in combating A. baumannii is that our understanding of the molecular mechanisms underlying the pathogenesis of A. baumannii is lacking. To identify potential virulence determinants of a contemporary multidrug-resistant isolate of A. baumannii, we used transposon insertion sequencing (TnSeq) of strain AB5075. A collection of 250,000 A. baumannii transposon mutants was analyzed for growth within Galleria mellonella larvae, an insect-based infection model. The screen identified 300 genes that were specifically required for survival and/or growth of A. baumannii inside G. mellonella larvae. These genes encompass both known, established virulence factors and several novel genes. Among these were more than 30 transcription factors required for growth in G. mellonella. A subset of the transcription factors was also found to be required for resistance to antibiotics and environmental stress. This work thus establishes a novel connection between virulence and resistance to both antibiotics and environmental stress in A. baumannii.

Importance: Acinetobacter baumannii is rapidly emerging as a significant human pathogen, largely because of disinfectant and antibiotic resistance, causing lethal infection in fragile hosts. Despite the increasing prevalence of infections with multidrug-resistant A. baumannii strains, little is known regarding not only the molecular mechanisms that allow A. baumannii to resist environmental stresses (i.e., antibiotics and disinfectants) but also how these pathogens survive within an infected host to cause disease. We employed a large-scale genetic screen to identify genes required for A. baumannii to survive and grow in an insect disease model. While we identified many known virulence factors harbored by A. baumannii, we also discovered many novel genes that likely play key roles in A. baumannii survival of exposure to antibiotics and other stress-inducing chemicals. These results suggest that selection for increased resistance to antibiotics and environmental stress may inadvertently select for increased virulence in A. baumannii.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.01660-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659468PMC
November 2015

Diverse protist grazers select for virulence-related traits in Legionella.

ISME J 2015 Jul 9;9(7):1607-18. Epub 2015 Jan 9.

Department of Microbiology, University of Chicago, Chicago, IL, USA.

It is generally accepted that selection for resistance to grazing by protists has contributed to the evolution of Legionella pneumophila as a pathogen. Grazing resistance is becoming more generally recognized as having an important role in the ecology and evolution of bacterial pathogenesis. However, selection for grazing resistance presupposes the existence of protist grazers that provide the selective pressure. To determine whether there are protists that graze on pathogenic Legionella species, we investigated the existence of such organisms in a variety of environmental samples. We isolated and characterized diverse protists that graze on L. pneumophila and determined the effects of adding L. pneumophila on the protist community structures in microcosms made from these environmental samples. Several unrelated organisms were able to graze efficiently on L. pneumophila. The community structures of all samples were markedly altered by the addition of L. pneumophila. Surprisingly, some of the Legionella grazers were closely related to species that are known hosts for L. pneumophila, indicating the presence of unknown specificity determinants for this interaction. These results provide the first direct support for the hypothesis that protist grazers exert selective pressure on Legionella to acquire and retain adaptations that contribute to survival, and that these properties are relevant to the ability of the bacteria to cause disease in people. We also report a novel mechanism of killing of amoebae by one Legionella species that requires an intact Type IV secretion system but does not involve intracellular replication. We refer to this phenomenon as 'food poisoning'.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ismej.2014.248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478701PMC
July 2015

Genetic Manipulation of Acinetobacter baumannii.

Curr Protoc Microbiol 2014 Nov 3;35:6G.2.1-11. Epub 2014 Nov 3.

Walter Reed Army Institute of Research, Department of Wound Infections, Silver Spring, Maryland.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen of clinical importance. A lack of genetic tools has hindered the research of this organism in the past; however, recently, various methods have been designed, modified, and optimized to facilitate the genetic manipulation of A. baumannii. This unit describes some of the recent genetic advances and new recombinant tools developed for this pathogen, including standard transformation and conjugation techniques specifically developed for the bacteria. As the need to understand the basic biology of A. baumannii increases with the prospect of developing new therapeutics, the use of the basic genetic methods herein can provide the critical first step to identify genes required for infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/9780471729259.mc06g02s35DOI Listing
November 2014

Host-directed antimicrobial drugs with broad-spectrum efficacy against intracellular bacterial pathogens.

mBio 2014 Jul 29;5(4):e01534-14. Epub 2014 Jul 29.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA.

We sought a new approach to treating infections by intracellular bacteria, namely, by altering host cell functions that support their growth. We screened a library of 640 Food and Drug Administration (FDA)-approved compounds for agents that render THP-1 cells resistant to infection by four intracellular pathogens. We identified numerous drugs that are not antibiotics but were highly effective in inhibiting intracellular bacterial growth with limited toxicity to host cells. These compounds are likely to target three kinds of host functions: (i) G protein-coupled receptors, (ii) intracellular calcium signals, and (iii) membrane cholesterol distribution. The compounds that targeted G protein receptor signaling and calcium fluxes broadly inhibited Coxiella burnetii, Legionella pneumophila, Brucella abortus, and Rickettsia conorii, while those directed against cholesterol traffic strongly attenuated the intracellular growth of C. burnetii and L. pneumophila. These pathways probably support intracellular pathogen growth so that drugs that perturb them may be therapeutic candidates. Combining host- and pathogen-directed treatments is a strategy to decrease the emergence of drug-resistant intracellular bacterial pathogens. Importance: Although antibiotic treatment is often successful, it is becoming clear that alternatives to conventional pathogen-directed therapy must be developed in the face of increasing antibiotic resistance. Moreover, the costs and timing associated with the development of novel antimicrobials make repurposed FDA-approved drugs attractive host-targeted therapeutics. This paper describes a novel approach of identifying such host-targeted therapeutics against intracellular bacterial pathogens. We identified several FDA-approved drugs that inhibit the growth of intracellular bacteria, thereby implicating host intracellular pathways presumably utilized by bacteria during infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.01534-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128363PMC
July 2014

Identification of novel Coxiella burnetii Icm/Dot effectors and genetic analysis of their involvement in modulating a mitogen-activated protein kinase pathway.

Infect Immun 2014 Sep 23;82(9):3740-52. Epub 2014 Jun 23.

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.01729-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187803PMC
September 2014

Analysis of the transcriptome of Legionella pneumophila hfq mutant reveals a new mobile genetic element.

Microbiology (Reading) 2013 Aug 31;159(Pt 8):1649-1660. Epub 2013 May 31.

Department of Natural Resource Sciences, Faculty of Agricultural and Environmental Sciences, McGill University, Ste-Anne-de-Bellevue, QC, Canada.

Hfq is a small RNA-binding protein involved in the post-transcriptional regulation of gene expression by affecting the stability of the mRNA and by mediating efficient pairing between small regulatory RNAs and their target mRNAs. In Legionella pneumophila, the aetiological agent of Legionnaires' disease, mutation of hfq results in increased duration of the lag phase and reduced growth in low-iron medium. In an effort to uncover genes potentially regulated by Hfq, the transcriptome of an hfq mutant strain was compared to that of the wild-type. Unexpectedly, many genes located within a 100 kb genomic island, including a section of the previously identified efflux island, were overexpressed in the hfq mutant strain. Since this island contains a putative conjugative system and an integrase, it was postulated that it could be a new integrated mobile genetic element. PCR analysis revealed that this region exists both as an integrated and as an episomal form in the cell population and that it undergoes differential excision in the hfq mutant background, which was further confirmed by trans-complementation of the hfq mutation. This new plasmid-like element was named pLP100. Differential excision did not affect the copy number of pLP100 at the population level. This region contains a copper efflux pump encoded by copA, and increased resistance to copper was observed for the hfq mutant strain that was abrogated in the complemented strain. A strain carrying a mutation of hfq and a deletion of the right side recombination site, attR, showed that overexpression of pLP100 genes and increased copper resistance in the hfq mutant strain were dependent upon excision of pLP100.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mic.0.067983-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972305PMC
August 2013

Legionella pneumophila transcriptional response following exposure to CuO nanoparticles.

Appl Environ Microbiol 2013 Apr 15;79(8):2713-20. Epub 2013 Feb 15.

U.S. EPA National Exposure Research Laboratory, Cincinnati, Ohio, USA.

Copper ions are an effective antimicrobial agent used to control Legionnaires' disease and Pontiac fever arising from institutional drinking water systems. Here, we present data on an alternative bactericidal agent, copper oxide nanoparticles (CuO-NPs), and its efficacy on Legionella pneumophila. In broth cultures, the CuO-NPs caused growth inhibition, which appeared to be concentration and exposure time dependent. The transcriptomic response of L. pneumophila to CuO-NP exposure was investigated by using a whole-genome microarray. The expression of genes involved in metabolism, transcription, translation, DNA replication and repair, and unknown/hypothetical proteins was significantly affected by exposure to CuO-NPs. In addition, expression of 21 virulence genes was also affected by exposure to CuO-NP and further evaluated by quantitative reverse transcription-PCR (qRT-PCR). Some virulence gene responses occurred immediately and transiently after addition of CuO-NPs to the cells and faded rapidly (icmV, icmW, lepA), while expression of other genes increased within 6 h (ceg29, legLC8, legP, lem19, lem24, lpg1689, and rtxA), 12 h (cegC1, dotA, enhC, htpX, icmE, pvcA, and sidF), and 24 h (legP, lem19, and ceg19), but for most of the genes tested, expression was reduced after 24 h of exposure. Genes like ceg29 and rtxA appeared to be the most responsive to CuO-NP exposures and along with other genes identified in this study may prove useful to monitor and manage the impact of drinking water disinfection on L. pneumophila.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.03462-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623207PMC
April 2013

Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal.

Proc Natl Acad Sci U S A 2013 Feb 4;110(8):E707-15. Epub 2013 Feb 4.

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.

Legionella and Coxiella are intracellular pathogens that use the virulence-related Icm/Dot type-IVB secretion system to translocate effector proteins into host cells during infection. These effectors were previously shown to contain a C-terminal secretion signal required for their translocation. In this research, we implemented a hidden semi-Markov model to characterize the amino acid composition of the signal, thus providing a comprehensive computational model for the secretion signal. This model accounts for dependencies among sites and captures spatial variation in amino acid composition along the secretion signal. To validate our model, we predicted and synthetically constructed an optimal secretion signal whose sequence is different from that of any known effector. We show that this signal efficiently translocates into host cells in an Icm/Dot-dependent manner. Additionally, we predicted in silico and experimentally examined the effects of mutations in the secretion signal, which provided innovative insights into its characteristics. Some effectors were found to lack a strong secretion signal according to our model. We demonstrated that these effectors were highly dependent on the IcmS-IcmW chaperons for their translocation, unlike effectors that harbor a strong secretion signal. Furthermore, our model is innovative because it enables searching ORFs for secretion signals on a genomic scale, which led to the identification and experimental validation of 20 effectors from Legionella pneumophila, Legionella longbeachae, and Coxiella burnetii. Our combined computational and experimental methodology is general and can be applied to the identification of a wide spectrum of protein features that lack sequence conservation but have similar amino acid characteristics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1215278110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581968PMC
February 2013

In vitro derivation of macrophage from guinea pig bone marrow with human M-CSF.

J Immunol Methods 2013 Mar 16;389(1-2):88-94. Epub 2013 Jan 16.

Section of Infectious Diseases, Department of Pediatrics, Comer Children's Hospital, University of Chicago Medical Center, Chicago, IL 60637, USA.

The guinea pig has a storied history as a model in the study of infectious disease and immunology. Because of reproducibility of data and availability of various reagents, inbred mice have since supplanted the guinea pig as the animal model-of-choice in these fields. However, several clinically-significant microorganisms do not cause the same pathology in mice, or mice may not be susceptible to these infections. These demonstrate the utility of other animal models - either as the primary method to study a particular infection, or to confirm or refute findings in the mouse before translating basic science into clinical practice. The mononuclear phagocyte, or macrophage (Mφ), plays a key role in antigen presentation and the pathogenesis of intracellular bacteria, such as Mycobacterium tuberculosis and Legionella pneumophila. Because of variable yield and difficult extraction from tissue, the preferred method of producing Mφ for in vitro studies is to expand murine bone marrow (BM) precursors with mouse macrophage colony-stimulating factor (M-CSF). This has not been shown in the guinea pig. Here, we report the empiric observation that human M-CSF - but not mouse M-CSF, nor human granulocyte/macrophage colony-stimulating factor - can be used to induce BM precursor differentiation into bonafide Mφ. The differentiated cells appeared as enlarged adherent cells, capable of both pinocytosis and large particle phagocytosis. Furthermore, we showed that these guinea pig BM-derived Mφ, similar to human monocyte/Mφ lines but unlike most murine BM Mφ, support growth of wild type L. pneumophila. This method may prove useful for in vitro studies of Mφ in the guinea pig, as well as in the translation of results found using mouse BM-derived Mφ towards studies in human immunology and infectious disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2013.01.005DOI Listing
March 2013

Methods to study legionella transcriptome in vitro and in vivo.

Methods Mol Biol 2013 ;954:567-82

Faculty of Agricultural and Environmental Sciences, Department of Natural Resource Sciences, McGill University, Quebec, Canada.

The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of virulence factors. Microarrays have been previously used to study gene regulation in Legionella pneumophila. In the past few years a sharp reduction of the costs associated with microarray experiments together with the availability of relatively inexpensive custom-designed commercial microarrays has made microarray technology an accessible tool for the majority of researchers. Here we describe the methodologies to conduct microarray experiments from in vitro and in vivo samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-62703-161-5_35DOI Listing
April 2013

A pathogen's journey in the host cell: Bridges between actin and traffic.

Bioarchitecture 2012 Feb;2(2):38-42

Manipulation of the actin cytoskeleton is a commonly used process by which bacterial pathogens and viruses are able to neutralize host defense mechanisms and subvert them in order to replicate in a hostile environment. Diverse bacteria display a wide array of mechanisms of regulation of microfilaments to enter, move within or exit the host cell. A less studied subject is how pathogens may co-opt the actin cytoskeleton to disturb vesicle trafficking pathways, namely phagolysosomal fusion, and avoid degradation. In fact, although actin plays a role in endosomal trafficking and phagosome maturation, the knowledge on the exact mechanisms and additional players is still scarce. Recently, we found that the Legionella pneumophila virulence factor VipA is an actin nucleator, associates with actin filaments and early endosomes during infection, and interferes in yeast organelle trafficking pathways, suggesting it may be linking actin dynamics to endosome biogenesis. Further studies on this protein, together with work on other bacterial effectors, may help shed light in the role of actin in endosomal maturation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383720PMC
http://dx.doi.org/10.4161/bioa.20422DOI Listing
February 2012

The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking.

PLoS Pathog 2012 Feb 23;8(2):e1002546. Epub 2012 Feb 23.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA.

Legionella pneumophila, the causative agent of Legionnaires' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1002546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285593PMC
February 2012

Whole-genome sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H.

J Bacteriol 2012 Mar;194(6):1613-4

Department of Microbiology, University of Chicago, Chicago, Illinois, USA.

We present the genomic sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H (ATCC 43290), a clinical isolate from the Colorado Department of Health, Denver, CO. This is the first example of a genome sequence of L. pneumophila from a serogroup other than serogroup 1. We highlight the similarities and differences relative to six genome sequences that have been reported for serogroup 1 strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.06626-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294838PMC
March 2012

Small Regulatory RNA and Legionella pneumophila.

Front Microbiol 2011 6;2:98. Epub 2011 May 6.

Complex Traits Group, Department of Microbiology, McGill University Montreal, QC, Canada.

Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires' disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2011.00098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153055PMC
November 2011

Legionella Pneumophila Transcriptome during Intracellular Multiplication in Human Macrophages.

Front Microbiol 2011 4;2:60. Epub 2011 Apr 4.

Department of Microbiology and Immunology, Columbia University Medical Center New York, NY, USA.

Legionella pneumophila is the causative agent of Legionnaires' disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called selective capture of transcribed sequences to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to l-arginine, l-histidine, and l-proline as well as many transport systems involved in amino acid and iron uptake. Genes involved in catabolism of glycerol are also induced during intracellular growth, suggesting that glycerol could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2011.00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128937PMC
October 2012

Chemical synthesis enables biochemical and antibacterial evaluation of streptolydigin antibiotics.

J Am Chem Soc 2011 Aug 20;133(31):12172-84. Epub 2011 Jul 20.

Department of Chemistry, University of Chicago, Chicago, Illinois 60607, USA.

Inhibition of bacterial transcription represents an effective and clinically validated anti-infective chemotherapeutic strategy. We describe the evolution of our approach to the streptolydigin class of antibiotics that target bacterial RNA polymerases (RNAPs). This effort resulted in the synthesis and biological evaluation of streptolydigin, streptolydiginone, streptolic acid, and a series of new streptolydigin-based agents. Subsequent biochemical evaluation of RNAP inhibition demonstrated that the presence of both streptolic acid and tetramic acid subunits was required for activity of this class of antibiotics. In addition, we identified 10,11-dihydrostreptolydigin as a new RNAP-targeting agent, which was assembled with high synthetic efficiency of 15 steps in the longest linear sequence. Dihydrostreptolydigin inhibited three representative bacterial RNAPs and displayed in vitro antibacterial activity against S. salivarius . The overall increase in synthetic efficiency combined with substantial antibacterial activity of this fully synthetic antibiotic demonstrates the power of organic synthesis in enabling design and comprehensive in vitro pharmacological evaluation of new chemical agents that target bacterial transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja2041964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174773PMC
August 2011

Cyclic diguanylate signaling proteins control intracellular growth of Legionella pneumophila.

mBio 2011 Jan 11;2(1):e00316-10. Epub 2011 Jan 11.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA.

Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires' disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00316-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023162PMC
January 2011

A new heterolobosean amoeba Solumitrus palustris n. g., n. sp. isolated from freshwater marsh soil.

J Eukaryot Microbiol 2011 Jan-Feb;58(1):60-7

Division of Biology, Lamont-Doherty Earth Observatory, Columbia University, Palisades, New York 10964, USA.

During the course of research on the bacterial feeding behavior and resistance of amoebae to virulent pathogens, we isolated a new strain of amoeba from organic rich soil at the margin of freshwater swamp in the northeastern United States. Light microscopic morphology is characteristically heterolobosean, resembling vahlkampfiids, including a broadened, limax shape, and eruptive locomotion, but occasionally becoming more contracted and less elongated with lateral or anterior bulges and somewhat branching sparse, uroidal filaments. Electron microscopic evidence, including mitochondria with flattened cristae surrounded by rough endoplasmic reticulum, further indicates a heterolobosean affinity. The solitary nucleus contains a centrally located nucleolus. Cysts are rounded with occasionally an eccentrically located nucleus. The cyst walls are relatively thin, becoming crenated, and loosely enclosing the cyst when mature. Molecular genetic evidence places this isolate among the Heterolobosea, branching most closely in a clade including Allovahlkampfia spelaea and previously isolated, un-named strains of soil amoebae. Based on differentiated features, including morphology of the uroid, cyst wall structure, and molecular genetic evidence that distinguish it from A. spelaea, a new genus and species, Solumitrus palustris, is proposed for this new heterolobosean.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1550-7408.2010.00520.xDOI Listing
April 2011

Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila.

J Bacteriol 2011 Mar 17;193(5):1114-21. Epub 2010 Dec 17.

Department of Microbiology and Immunology, Columbia University, New York, New York 100321, USA.

Natural transformation by competence is a major mechanism of horizontal gene transfer in bacteria. Competence is defined as the genetically programmed physiological state that enables bacteria to actively take up DNA from the environment. The conditions that signal competence development are multiple and elusive, complicating the understanding of its evolutionary significance. We used expression of the competence gene comEA as a reporter of competence development and screened several hundred molecules for their ability to induce competence in the freshwater living pathogen Legionella pneumophila. We found that comEA expression is induced by chronic exposure to genotoxic molecules such as mitomycin C and antibiotics of the fluoroquinolone family. These results indicated that, in L. pneumophila, competence may be a response to genotoxic stress. Sunlight-emitted UV light represents a major source of genotoxic stress in the environment and we found that exposure to UV radiation effectively induces competence development. For the first time, we show that genetic exchanges by natural transformation occur within an UV-stressed population. Genotoxic stress induces the RecA-dependent SOS response in many bacteria. However, genetic and phenotypic evidence suggest that L. pneumophila lacks a prototypic SOS response and competence development in response to genotoxic stress is RecA independent. Our results strengthen the hypothesis that competence may have evolved as a DNA damage response in SOS-deficient bacteria. This parasexual response to DNA damage may have enabled L. pneumophila to acquire and propagate foreign genes, contributing to the emergence of this human pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.01146-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067580PMC
March 2011

ArgR-regulated genes are derepressed in the Legionella-containing vacuole.

J Bacteriol 2010 Sep 9;192(17):4504-16. Epub 2010 Jul 9.

Department of Microbiology & Immunology, Columbia University Medical Center, New York, New York 10032, USA.

Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. L-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of L-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of L-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that L-arginine availability functions as a regulatory signal during Legionella intracellular growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00465-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937375PMC
September 2010

Legionella pneumophila 6S RNA optimizes intracellular multiplication.

Proc Natl Acad Sci U S A 2010 Apr 5;107(16):7533-8. Epub 2010 Apr 5.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA.

Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which 6 were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed in postexponential phase, and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of sigma(70)-containing RNA polymerase. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA, as well as many genes involved in acquisition of nutrients and genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0911764107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867745PMC
April 2010
-->