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Gary K Schoolnik

Publications and source records attributed to Gary K Schoolnik.

At least 19 recordsLinked to original sources
PubMed2006

The competitive cost of antibiotic resistance in Mycobacterium tuberculosis.

Mathematical models predict that the future of the multidrug-resistant tuberculosis epidemic will depend on the fitness cost of drug resistance. We show that in laboratory-derived mutants of Mycobacterium tuberculosis, rifampin resistance is universally associated with a competitive fitness cost and that this cost is determined by the specific resistance mutation and strain genetic background. In contrast, we demonstrate that prolonged patient treatment can result in multidrug-resistant strains with no fitness defect and that strains with low- or no-cost resistance mutations are also the most frequent among clinical isolates.

Amino Acid Substitution↗
PubMed2006

Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis.

The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-gamma) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-gamma response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-gamma responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-gamma responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.

Adolescent↗
PubMed2006

Evidence supporting predicted metabolic pathways for Vibrio cholerae: gene expression data and clinical tests.

Vibrio cholerae, the etiological agent of the diarrheal illness cholera, can kill an infected adult in 24 h. V.cholerae lives as an autochthonous microbe in estuaries, rivers and coastal waters. A better understanding of its metabolic pathways will assist the development of more effective treatments and will provide a deeper understanding of how this bacterium persists in natural aquatic habitats. Using the completed V.cholerae genome sequence and PathoLogic software, we created VchoCyc, a pathway-genome database that predicted 171 likely metabolic pathways in the bacterium. We report here experimental evidence supporting the computationally predicted pathways. The evidence comes from microarray gene expression studies of V.cholerae in the stools of three cholera patients [D. S. Merrell, S. M. Butler, F. Qadri, N. A. Dolganov, A. Alam, M. B. Cohen, S. B. Calderwood, G. K. Schoolnik and A. Camilli (2002) Nature, 417, 642-645.], from gene expression studies in minimal growth conditions and LB rich medium, and from clinical tests that identify V.cholerae. Expression data provide evidence supporting 92 (53%) of the 171 pathways. The clinical tests provide evidence supporting seven pathways, with six pathways supported by both methods. VchoCyc provides biologists with a useful tool for analyzing this organism's metabolic and genomic information, which could lead to potential insights into new anti-bacterial agents. VchoCyc is available in the BioCyc database collection (http://BioCyc.org).

Bacteriological Techniques↗
PubMed2006

Expression profiling of host pathogen interactions: how Mycobacterium tuberculosis and the macrophage adapt to one another.

It has recently become feasible to quantify all mRNAs encoded by the genomes of bacterial pathogens and their eukaryotic host cells and to apply this approach to study the interaction of Mycobacterium tuberculosis with its primary host cell, the macrophage. These studies helped to identify regulatory circuits which mediate adaptation of the M. tuberculosis transcriptome to intraphagosomal environments and stimulated hypotheses for the function of these circuits in human tuberculosis. The macrophage transcriptome reacts to infections with the induction of a pathogen-unspecific expression program as well as the induction of pathogen-specific expression signatures, both of which contribute to the immunologic activation of the infected cell. M. tuberculosis induced changes in the macrophage transcriptome are mediated by Toll-like receptor dependent and Toll-like receptor independent signal transduction pathways. This response is shaped by macrophage produced reactive nitrogen and oxygen molecules and affected by viability and virulence of the pathogen.

Animals↗
PubMed2006

Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae.

Phase variation between smooth and rugose colony variants of Vibrio cholerae is predicted to be important for the pathogen's survival in its natural aquatic ecosystems. The rugose variant forms corrugated colonies, exhibits increased levels of resistance to osmotic, acid, and oxidative stresses, and has an enhanced capacity to form biofilms. Many of these phenotypes are mediated in part by increased production of an exopolysaccharide termed VPS. In this study, we compared total protein profiles of the smooth and rugose variants using two-dimensional gel electrophoresis and identified one protein that is present at a higher level in the rugose variant. A mutation in the gene encoding this protein, which does not have any known homologs in the protein databases, causes cells to form biofilms that are more fragile and sensitive to sodium dodecyl sulfate than wild-type biofilms. The results indicate that the gene, termed rbmA (rugosity and biofilm structure modulator A), is required for rugose colony formation and biofilm structure integrity in V. cholerae. Transcription of rbmA is positively regulated by the response regulator VpsR but not VpsT.

Bacterial Proteins↗
PubMed2006

RpoS controls the Vibrio cholerae mucosal escape response.

Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.

Animals↗
PubMed2005

Chitin induces natural competence in Vibrio cholerae.

The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.

Animals↗
PubMed2004

The Vibrio cholerae chitin utilization program.

Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms. Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc)(2-6), GlcNAc, or the glucosamine dimer (GlcN)2 identified three sets of differentially regulated genes. We show that (i) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc)(2-6) gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP (chitin-regulated pilus) that confers a significant growth advantage to V. cholerae on a chitin surface; (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; (iii) (GlcN)2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and (iv) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry. Collectively, these results provide a global portrait of a complex, multistage V. cholerae program for the efficient utilization of chitin.

Cell Adhesion↗
PubMed2004

Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant.

Reversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the rugose and smooth variants of the same strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPS(El Tor), a rugose-associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation-specific gene expression. Bioinformatics analysis of these expression data shows that 'rugosity' and 'smoothness' are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.

Adaptation, Physiological↗
PubMed2003

Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4.

The predominant mode of HIV transmission worldwide is via heterosexual contact, with the cervico-vaginal mucosa being the main portal of entry in women. The cervico-vaginal mucosa is naturally colonized with commensal bacteria, primarily lactobacilli. To address the urgent need for female-controlled approaches to block the heterosexual transmission of HIV, we have engineered natural human vaginal isolates of Lactobacillus jensenii to secrete two-domain CD4 (2D CD4) proteins. The secreted 2D CD4 recognized a conformation-dependent anti-CD4 antibody and bound HIV type 1 (HIV-1) gp120, suggesting that the expressed proteins adopted a native conformation. Single-cycle infection assays using HIV-1HxB2 carrying a luciferase reporter gene demonstrated that Lactobacillus-derived 2D CD4 inhibited HIV-1 entry into target cells in a dose-dependent manner. Importantly, coincubation of the engineered bacteria with recombinant HIV-1HxB2 reporter virus led to a significant decrease in virus infectivity of HeLa cells expressing CD4-CXCR4-CCR5. Engineered lactobacilli also caused a modest, but statistically significant, decrease in infectivity of a primary isolate, HIV-1JR-FL. This represents an important first step toward the development of engineered commensal bacteria within the vaginal microflora to inhibit heterosexual transmission of HIV.

Antiviral Agents↗
PubMed2003

Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program.

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.

Mycobacterium tuberculosis↗
PubMed2003

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon gamma- and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and beta-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of sigmaE-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.

Animals↗
PubMed2003

Microarray expression profiling: capturing a genome-wide portrait of the transcriptome.

The bacterial transcriptome is a dynamic entity that reflects the organism's immediate, ongoing and genome-wide response to its environment. Microarray expression profiling provides a comprehensive portrait of the transcriptional world enabling us to view the organism as a 'system' that is more than the sum of its parts. The vigilance of microorganisms to environmental change, the alacrity of the transcriptional response, the short half-life of bacterial mRNA and the genome-scale nature of the investigation collectively explain the power of this method. These same features pose the most significant experimental design and execution issues which, unless surmounted, predictably generate a distorted image of the transcriptome. Conversely, the expression profile of a properly conceived and conducted microarray experiment can be used for hypothesis testing: disclosure of the metabolic and biosynthetic pathways that underlie adaptation of the organism to chang-ing conditions of growth; the identification of co-ordinately regulated genes; the regulatory circuits and signal transduction systems that mediate the adaptive response; and temporal features of developmental programmes. The study of bacterial pathogenesis by microarray expression profiling poses special challenges and opportunities. Although the technical hurdles are many, obtaining expression profiles of an organism growing in tissue will probably reveal strategies for growth and survival in the host's microenvironment. Identifying these colonization strategies and their cognate expression patterns involves a 'deconstruction' process that combines bioinformatics analysis and in vitro DNA array experimentation.

Bacteria↗
PubMed2003

Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis.

Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.

Aspartic Acid↗
PubMed2003

Structural and topographical studies of the type IV bundle-forming pilus assembly complex of enteropathogenic Escherichia coli.

The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes. BFP filament biogenesis and function are encoded by the 14-gene bfp operon. The BFP assembly complex, containing a BfpB-His6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography. Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex. Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space. A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.

Cell Polarity↗
PubMed2002

Host-induced epidemic spread of the cholera bacterium.

The factors that enhance the transmission of pathogens during epidemic spread are ill defined. Water-borne spread of the diarrhoeal disease cholera occurs rapidly in nature, whereas infection of human volunteers with bacteria grown in vitro is difficult in the absence of stomach acid buffering. It is unclear, however, whether stomach acidity is a principal factor contributing to epidemic spread. Here we report that characterization of Vibrio cholerae from human stools supports a model whereby human colonization creates a hyperinfectious bacterial state that is maintained after dissemination and that may contribute to epidemic spread of cholera. Transcriptional profiling of V. cholerae from stool samples revealed a unique physiological and behavioural state characterized by high expression levels of genes required for nutrient acquisition and motility, and low expression levels of genes required for bacterial chemotaxis.

Animals↗
PubMed2002

Microarray analysis of bacterial pathogenicity.

The DNA microarray, a surface that contains an ordered arrangement of each identified open reading frame of a sequenced genome, is the engine of functional genomics. Its output, the expression profile, provides a genome wide snap-shot of the transcriptome. Refined by array-specific statistical instruments and data-mined by clustering algorithms and metabolic pathway databases, the expression profile discloses, at the transcriptional level, how the microbe adapts to new conditions of growth--the regulatory networks that govern the adaptive response and the metabolic and biosynthetic pathways that effect the new phenotype. Adaptation to host microenvironments underlies the capacity of infectious agents to persist in and damage host tissues. While monitoring the whole genome transcriptional response of bacterial pathogens within infected tissues has not been achieved, it is likely that the complex, tissue-specific response is but the sum of individual responses of the bacteria to specific physicochemical features that characterize the host milieu. These are amenable to experimentation in vitro and whole-genome expression studies of this kind have defined the transcriptional response to iron starvation, low oxygen, acid pH, quorum-sensing pheromones and reactive oxygen intermediates. These have disclosed new information about even well-studied processes and provide a portrait of the adapting bacterium as a 'system', rather than the product of a few genes or even a few regulons. Amongst the regulated genes that compose this adaptive system are transcription factors. Expression profiling experiments of transcription factor mutants delineate the corresponding regulatory cascade. The genetic basis for pathogenicity can also be studied by using microarray-based comparative genomics to characterize and quantify the extent of genetic variability within natural populations at the gene level of resolution. Also identified are differences between pathogen and commensal that point to possible virulence determinants or disclose evolutionary history. The host vigorously engages the pathogen; expression studies using host genome microarrays and bacterially infected cell cultures show that the initial host reaction is dominated by the innate immune response. However, within the complex expression profile of the host cell are components mediated by pathogen-specific determinants. In the future, the combined use of bacterial and host microarrays to study the same infected tissue will reveal the dialogue between pathogen and host in a gene-by-gene and site- and time-specific manner. Translating this conversation will not be easy and will probably require a combination of powerful bioinformatic tools and traditional experimental approaches--and considerable effort and time.

Animals↗