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Howard A Shuman

Publications and source records attributed to Howard A Shuman.

11 recordsLinked to original sources
PubMed2005

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

Bacterial Proteins↗
PubMed2005

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii.

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

Acanthamoeba castellanii↗
PubMed2005

Evidence for acquisition of Legionella type IV secretion substrates via interdomain horizontal gene transfer.

Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the gamma-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.

Amino Acid Motifs↗
PubMed2004

The genomic sequence of the accidental pathogen Legionella pneumophila.

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

DNA, Bacterial↗
PubMed2004

Legionella effectors that promote nonlytic release from protozoa.

Legionella pneumophila, the bacterial agent of legionnaires' disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot-dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen.

Acanthamoeba↗
PubMed2004

Comparative sequence analysis of the icm/dot genes in Legionella.

The icm/dot genes in Legionella pneumophila are essential for the ability of the bacteria to survive within macrophages in lung infections such as Legionnaires' disease, or amoebae in nature. The 22 genes of the complex, thought to encode a transport apparatus for transfer of effector molecules into the host cell cytoplasm, are located in two chromosomal loci. We demonstrate that these genes are present in all the L. pneumophila strains examined herein, but display a wide range of sequence variation among the different strains, none of which are clearly associated with virulence potential. The strains fall within seven phylogenetic groups, but discrepancies among the gene trees indicate a complicated evolutionary history for the icm/dot loci, with perhaps two independent gene acquisition events and subsequent genomic rearrangements. Significant findings include a probable t-SNARE domain in IcmG that may indicate a direct role for this putative inner membrane protein in altering the host's membrane fusion machinery, a potential functional domain in the central hydrophobic portion of IcmK that may allow it to participate in forming the pore of the secretion complex, and strict conservation of the amino acid physicochemical characteristics in the IcmP region corresponding to the trbA domain that could play a role in molecular transfer.

Amino Acid Sequence↗
PubMed2004

Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum.

The Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum. Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically co-localize with DsRed-labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.

Animals↗
PubMed2003

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex.

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.

ATP-Binding Cassette Transporters↗
PubMed2003

The art and design of genetic screens: Escherichia coli.

This article summarizes the general principles of selections and screens in Escherichia coli. The focus is on the lac operon, owing to its inherent simplicity and versatility. Examples of different strategies for mutagenesis and mutant discovery are described. In particular, the usefulness and effectiveness of simple colour-based screens are illustrated. The power of lac genetics can be applied to almost any bacterial system with gene fusions that hook any gene of interest to lacZ, which is the structural gene that encodes beta-galactosidase. The diversity of biological processes that can be studied with lac genetics is remarkable and includes DNA metabolism, gene regulation and signal transduction, protein localization and folding, and even electron transport.

DNA Transposable Elements↗
PubMed2003

Virulence conversion of Legionella pneumophila by conjugal transfer of chromosomal DNA.

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x 10(9) CFU) and Chicago-2S (approximately 8.9 x 10(9) CFU) were mated typically yielded 10(3) Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.

Acanthamoeba↗