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Exit Mechanisms of the Intracellular Bacterium Ehrlichia  [PDF]
Sunil Thomas,Vsevolod L. Popov,David H. Walker
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015775
Abstract: The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the causative agent of human monocytotropic ehrlichiosis. Ehrlichia muris and Ixodes ovatus Ehrlichia (IOE) are agents of mouse models of ehrlichiosis. The mechanism by which Ehrlichia are transported from an infected host cell to a non-infected cell has not been demonstrated.
SNARE Protein Mimicry by an Intracellular Bacterium  [PDF]
Cédric Delevoye,Michael Nilges,Pierre Dehoux,Fabienne Paumet,Stéphanie Perrinet,Alice Dautry-Varsat,Agathe Subtil
PLOS Pathogens , 2008, DOI: 10.1371/journal.ppat.1000022
Abstract: Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.
Analysis of Convergent Gene Transcripts in the Obligate Intracellular Bacterium Rickettsia prowazekii  [PDF]
Andrew Woodard,David O. Wood
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016537
Abstract: Termination of transcription is an important component of bacterial gene expression. However, little is known concerning this process in the obligate intracellular pathogen and model for reductive evolution, Rickettsia prowazekii. To assess transcriptional termination in this bacterium, transcripts of convergent gene pairs, some containing predicted intrinsic terminators, were analyzed. These analyses revealed that, rather than terminating at a specific site within the intervening region between the convergent genes, most of the transcripts demonstrated either a lack of termination within this region, which generated antisense RNA, or a putative non-site-specific termination that occurred throughout the intervening sequence. Transcripts terminating at predicted intrinsic terminators, as well as at a putative Rho-dependant terminator, were also examined and found to vary based on the rickettsial host environment. These results suggest that transcriptional termination, or lack thereof, plays a role in rickettsial gene regulation.
Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity  [PDF]
Rodolfo D. Vicetti Miguel, Stephen A. K. Harvey, William A. LaFramboise, Seth D. Reighard, Dean B. Matthews, Thomas L. Cherpes
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0058565
Abstract: While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4+ T cells. Also among women with genital tract Chlamydia infection, peripheral CD3+ CD4+ and CD3+ CD4- cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-γ, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures.
The Intracellular Citrus Huanglongbing Bacterium, ‘Candidatus Liberibacter asiaticus’ Encodes Two Novel Autotransporters  [PDF]
Guixia Hao, Michael Boyle, Lijuan Zhou, Yongping Duan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0068921
Abstract: Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of ‘Candidatus Liberibacter asiaticus’ (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.
Asymmetry in the presence of migration stabilizes multistrain disease outbreaks  [PDF]
Simone Bianco,Leah B. Shaw
Quantitative Biology , 2009,
Abstract: We study the effect of migration between coupled populations, or patches, on the stability properties of multistrain disease dynamics. The epidemic model used in this work displays a Hopf bifurcation to oscillations in a single well mixed population. It is shown numerically that migration between two non-identical patches stabilizes the endemic steady state, delaying the onset of large amplitude outbreaks and reducing the total number of infections. This result is motivated by analyzing generic Hopf bifurcations with different frequencies and with diffusive coupling between them. Stabilization of the steady state is again seen, indicating that our observation in the full multistrain model is based on qualitative characteristics of the dynamics rather than on details of the disease model.
A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis
Marnix H Medema, Miaomiao Zhou, Sacha AFT van Hijum, Jolein Gloerich, Hans JCT Wessels, Roland J Siezen, Marc Strous
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-299
Abstract: Two training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive.A physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K. stuttgartiensis. This result provides strong in silico support for the existing experimental evidence for the existence of an organelle in this bacterium, and is an important step forward in unravelling a geochemically relevant case of cytoplasmic differentiation in bacteria. The predicted dual location of the Sec-translocation system and the apparent absence of a specific N- or C-terminal signal in the organellar proteins suggests that additional chaperones may be necessary that act on an as-yet unknown property of the targeted proteins.Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into nitrogen and are major players in the biogeochemical nitrogen cycle [1-4]. They comprise a monophyletic taxon within the Planctomycetes phylum. Like other Planctomycetes, they possess an unusual cel
Genome-wide screen for temperature-regulated genes of the obligate intracellular bacterium, Rickettsia typhi
Sheila M Dreher-Lesnick, Shane M Ceraul, M Sayeedur Rahman, Abdu F Azad
BMC Microbiology , 2008, DOI: 10.1186/1471-2180-8-61
Abstract: Temperature-responsive genes belonged to multiple functional categories including among others, transcription, translation, posttranslational modification/protein turnover/chaperones and intracellular trafficking and secretion. A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database. The microarray results were validated with quantitative real time RT-PCR.This microarray screen identified various genes that were differentially expressed upon a shift in temperature from 37°C to 25°C. Further characterization of the identified genes may provide new insights into the ability of R. typhi to successfully transition between its mammalian and arthropod hosts.Rickettsiae are obligate intracellular bacteria, and are best known as the arthropod-borne disease agents of spotted and typhus fevers in humans. These diseases are prevalent throughout the world, and continue to pose potential public health problems [1]. Rickettsia typhi is the etiologic agent of murine typhus, a reemerging febrile illness that is endemic in coastal areas throughout the world. Its reemergence and associated epidemic outbreaks are attributed to changes in the environment and human behavior [2]. Current research efforts have focused on molecular interactions between pathogenic rickettsiae and their mammalian hosts. Studies examining arthropod-rickettsiae interactions are more scarce, despite their importance for understanding the maintenance of rickettsiae in nature [1].Rickettsial homeostasis is continuously regulated as rickettsiae cycle between the warm-blooded vertebrate and poikilothermic invertebrate hosts. The effect of environmental temperature on gene expression is well documented for other arthropod-borne pathogens such as the agents of plague and Lyme disease, Yersinia pestis and Borrelia burgdorferi, respectively. For example, gene and protein expression studies have revealed that Y. pestis transmission
Complete genome of Phenylobacterium zucineum – a novel facultative intracellular bacterium isolated from human erythroleukemia cell line K562
Yingfeng Luo, Xiaoli Xu, Zonghui Ding, Zhen Liu, Bing Zhang, Zhiyu Yan, Jie Sun, Songnian Hu, Xun Hu
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-386
Abstract: Here, we report the whole genome sequence of the type strain HLK1T. The genome consists of a circular chromosome (3,996,255 bp) and a circular plasmid (382,976 bp). It encodes 3,861 putative proteins, 42 tRNAs, and a 16S-23S-5S rRNA operon. Comparative genomic analysis revealed that it is phylogenetically closest to Caulobacter crescentus, a model species for cell cycle research. Notably, P. zucineum has a gene that is strikingly similar, both structurally and functionally, to the cell cycle master regulator CtrA of C. crescentus, and most of the genes directly regulated by CtrA in the latter have orthologs in the former.This work presents the first complete bacterial genome in the genus Phenylobacterium. Comparative genomic analysis indicated that the CtrA regulon is well conserved between C. crescentus and P. zucineum.Phenylobacterium zucineum strain HLK1T is a facultative intracellular microbe recently identified by us [1]. It is a rod-shaped Gram-negative bacterium 0.3–0.5 × 0.5–2 μm in size. It belongs to the genus Phenylobacterium [2], which presently comprises 5 species, P. lituiforme (FaiI3T) [3], P. falsum (AC49T) [4], P. immobile (ET) [2], P. koreense (Slu-01T) [5], and P. zucineum (HLK1T) [1]. They were isolated from subsurface aquifer, alkaline groundwater, soil, activated sludge from a wastewater treatment plant, and the human leukemia cell line K562, respectively. Except for P. zucineum, they are environmental bacteria, and there is no evidence that these microbes are associated with eukaryotic cells. The HLK1T strain, therefore, represents the only species so far in the genus Phenylobacterium that can infect and survive in human cells. Since most, if not all, of the known microbes that can invade human cells are pathogenic, we proposed that HLK1T may have pathogenic relevance to humans [1]. Unlike the known intracellular pathogens that undergo a cycle involving invasion, overgrowth, and disruption of the host cells, and repeating the cycle by invading
Genetic diversity of the obligate intracellular bacterium Chlamydophila pneumoniae by genome-wide analysis of single nucleotide polymorphisms: evidence for highly clonal population structure
Thomas Rattei, Stephan Ott, Michaela Gutacker, Jan Rupp, Matthias Maass, Stefan Schreiber, Werner Solbach, Thierry Wirth, Jens Gieffers
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-355
Abstract: We studied the genetic diversity of C. pneumoniae by analysing synonymous single nucleotide polymorphisms (sSNPs) that are under reduced selection pressure. We conducted an in silico analysis of the four sequenced genomes, chose 232 representative sSNPs and analysed the loci in 38 C. pneumoniae isolates. We identified 15 different genotypes that were separated in four major clusters. Clusters were not associated with anatomical or geographical origin. However, animal lineages are basal on the C. pneumomiae phylogeny, suggesting a recent transmission to humans through successive bottlenecks some 150,000 years ago. A lack of detectable variation in 17 isolates emphasizes the extraordinary genetic conservation of this species and the high clonality of the population. Moreover, the largest cluster, which encompasses 80% of all analysed strains, is an extremely young clade, that went through an important population expansion some 3,300 years ago.sSNPs have proven useful as a sensitive marker to gain new insights into genetic diversity, population structure and evolutionary history of C. pneumoniae.The order Chlamydiales evolved from their free-living ancestors about 500–1000 million years ago (mya) [1], establishing their intracellular life in lower eukaryotes. Their life cycle consists of infectious elementary bodies and intracellularly replicative reticulate bodies (for recent review see e.g. [2]. The divergence of C. trachomatis and C. pneumoniae 50–200 mya proceeds the appearance of Homo sapiens and, thus, each lineage possessed sufficient biological potential to exploit new hosts [1] Gene acquisition by horizontal gene transfer (HGT) was not a driving force in the evolution of the Chlamydiales; less than 1 % of the total gene number was estimated to result from HGT [3]. Instead, while adapting to the homeostatic niche within their specific hosts, all Chlamydiaceae species reduced their genome size to little more than 1 Mb. At least 80 % of the genes in any sequenced
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