Genome-wide screen for temperature-regulated genes of the obligate intracellular bacterium, Rickettsia typhi

<p>Abstract</p> <p>Background</p> <p>The ability of rickettsiae to survive in multiple eukaryotic host environments provides a good model for studying pathogen-host molecular interactions. <it>Rickettsia typhi</it>, the etiologic agent of murine typhus, is a strictly intracellular gram negative α-proteobacterium, which is transmitted to humans by its arthropod vector, the oriental rat flea, <it>Xenopsylla cheopis</it>. Thus, <it>R. typhi </it>must cycle between mammalian and flea hosts, two drastically different environments. We hypothesize that temperature plays a role in regulating host-specific gene expression, allowing <it>R. typhi </it>to survive in mammalian and arthropod hosts. In this study, we used Affymetrix microarrays to screen for temperature-induced genes upon a temperature shift from 37°C to 25°C, mimicking the two different host temperatures <it>in vitro</it>.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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 <it>R. typhi </it>to successfully transition between its mammalian and arthropod hosts.</p

An Anomalous Type IV Secretion System in Rickettsia Is Evolutionarily Conserved

Bacterial type IV secretion systems (T4SSs) comprise a diverse transporter family functioning in conjugation, competence, and effector molecule (DNA and/or protein) translocation. Thirteen genome sequences from Rickettsia, obligate intracellular symbionts/pathogens of a wide range of eukaryotes, have revealed a reduced T4SS relative to the Agrobacterium tumefaciens archetype (vir). However, the Rickettsia T4SS has not been functionally characterized for its role in symbiosis/virulence, and none of its substrates are known.Superimposition of T4SS structural/functional information over previously identified Rickettsia components implicate a functional Rickettsia T4SS. virB4, virB8 and virB9 are duplicated, yet only one copy of each has the conserved features of similar genes in other T4SSs. An extraordinarily duplicated VirB6 gene encodes five hydrophobic proteins conserved only in a short region known to be involved in DNA transfer in A. tumefaciens. virB1, virB2 and virB7 are newly identified, revealing a Rickettsia T4SS lacking only virB5 relative to the vir archetype. Phylogeny estimation suggests vertical inheritance of all components, despite gene rearrangements into an archipelago of five islets. Similarities of Rickettsia VirB7/VirB9 to ComB7/ComB9 proteins of epsilon-proteobacteria, as well as phylogenetic affinities to the Legionella lvh T4SS, imply the Rickettsiales ancestor acquired a vir-like locus from distantly related bacteria, perhaps while residing in a protozoan host. Modern modifications of these systems likely reflect diversification with various eukaryotic host cells.We present the rvh (Rickettsiales vir homolog) T4SS, an evolutionary conserved transporter with an unknown role in rickettsial biology. This work lays the foundation for future laboratory characterization of this system, and also identifies the Legionella lvh T4SS as a suitable genetic model

Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis▿

Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPI's anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPI's trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the tick's defense response to rickettsiae

New Tick Defensin Isoform and Antimicrobial Gene Expression in Response to Rickettsia montanensis Challenge

Recent studies aimed at elucidating the rickettsia-tick interaction have discovered that the spotted fever group rickettsia Rickettsia montanensis, a relative of R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, induces differential gene expression patterns in the ovaries of the hard tick Dermacentor variabilis. Here we describe a new defensin isoform, defensin-2, and the expression patterns of genes for three antimicrobials, defensin-1 (vsnA1), defensin-2, and lysozyme, in the midguts and fat bodies of D. variabilis ticks that were challenged with R. montanensis. Bioinformatic and phylogenetic analyses of the primary structure of defensin-2 support its role as an antimicrobial. The tissue distributions of the three antimicrobials, especially the two D. variabilis defensin isoforms, are markedly different, illustrating the immunocompetence of the many tissues that R. montanensis presumably invades once acquired by the tick. Antimicrobial gene expression patterns in R. montanensis-challenged ticks suggest that antimicrobial genes play a role during the acquisition-invasion stages in the tick

The lspA Gene, Encoding the Type II Signal Peptidase of Rickettsia typhi: Transcriptional and Functional Analysis

Lipoprotein processing by the type II signal peptidase (SPase II) is known to be critical for intracellular growth and virulence for many bacteria, but its role in rickettsiae is unknown. Here, we describe the analysis of lspA, encoding a putative SPase II, an essential component of lipoprotein processing in gram-negative bacteria, from Rickettsia typhi. Alignment of deduced amino acid sequences shows the presence of highly conserved residues and domains that are essential for SPase II activity in lipoprotein processing. The transcription of lspA, lgt (encoding prolipoprotein transferase), and lepB (encoding type I signal peptidase), monitored by real-time quantitative reverse transcription-PCR, reveals a differential expression pattern during various stages of rickettsial intracellular growth. The higher transcriptional level of all three genes at the preinfection time point indicates that only live and metabolically active rickettsiae are capable of infection and inducing host cell phagocytosis. lspA and lgt, which are involved in lipoprotein processing, show similar levels of expression. However, lepB, which is involved in nonlipoprotein secretion, shows a higher level of expression, suggesting that LepB is the major signal peptidase for protein secretion and supporting our in silico prediction that out of 89 secretory proteins, only 14 are lipoproteins. Overexpression of R. typhi lspA in Escherichia coli confers increased globomycin resistance, indicating its function as SPase II. In genetic complementation, recombinant lspA from R. typhi significantly restores the growth of temperature-sensitive E. coli Y815 at the nonpermissive temperature, supporting its biological activity as SPase II in prolipoprotein processing

Additional file 1 of Nanobodies as potential tools for microbiological testing of live biotherapeutic products

Additional file 1:Table S1. Bacterial strains, plasmids, and primers used in this study. Figure S1. ELISA results of the interaction between different lactobacilli and secreted nanobodies. (A) Lc58 and Lc38 nanobody interaction between target L. crispatus antigen (strains 33820 and 33197) and control lactobacilli antigens. (B) Lj94 and Lj75 nanobody interaction between target L. jensenii JV-V16 antigen and control lactobacilli antigens. Secreted nanobody concentrations were evaluated using Octet (Sartorius) and the preparations were diluted to 1 μg/ml for experiments. Figure S2. SDS PAGE gels showing purified nanobodies and fluorescently tagged nanobodies. Proteins were loaded on NuPAGE 4–12% BisTris gels and stained with Coomasie Blue. The expected molecular mass of each protein and the lane in which the purified protein was run is indicated in the boxes below the gels. Molecular weight markers are identified on the left. Please note that under boiling SDS conditions, TagRFP is known to fragment. The additional bands observed in (B) lane C are likely due to the fragmentation of sample preparation for SDS PAGE. Figure S3. L. jensenii 115-3-CHN Lj75 antigen identification. (A) AA sequence analysis of (1) the originally annotated AA sequence of L. jensenii 115-3-CHN antigen (EEX23860.1), (2) the confirmed L. jensenii JV-V16 Lj75 antigen AA sequence, and (3) the extended L. jensenii 115-3-CHN Lj75 antigen AA sequence. Green above sequence analysis indicates 100% AA sequence identity. (B) Depiction of unique peptide hits along the AA sequence of the corrected L. jensenii 115-3-CHN antigen sequence. Green indicates where in the AA sequence the unique peptides match. Figure S4. L. crispatus strain lysate western blots with Lc58. L. crispatus EX8 VC07 (Lane 1), L. crispatus 125-2-CHN (Lane 2), or L. jensenii 25258 (lane 3) lysates were probed with Lc58. Lc58 binding was detected with an anti-his HRP conjugated secondary antibody. Figure S5. Detection of nanobody target candidate expression by western blot with HRP conjugated anti-FLAG antibody probing. (A) Lc58 target candidates; Lane 1, L. crispatus 125-2-CHN lysate; Lane 2, empty vector; Lane 3, S-layer (EEU18441.1) ; Lane 4, Bacterial Ig-domain protein (EEU19392.1) ; Lane 5, Cell separation protein (EEU18637.1). (B) Lj75 candidates; Lane 1, L. jensenii JV-V16; Lane 2, empty vector; Lane 3, NlPC/P60 family protein (EFH30000.1); Lane 4, Hypothetical protein (EFH30544.1).Figure S6. Use of SYTOX Green Ready Flow reagent to distinguish live from dead cells. SYTOX (ThermoFisher) is a cell impermeant nucleic acid stain that enters cells with damaged membranes and binds nucleic acids. (A) Untreated and unstained L. crispatus 33820, (B) Untreated L. crispatus 33820 solution (prepared same as flow cytometry samples), and (C) Isopropyl alcohol treated (70%, 25 min)L. crispatus 33820. (D) Untreated and unstained L. jensenii 115-3-CHN, (B) Untreated L. jensenii 115-3-CHN solution (prepared same as flow cytometry samples), and (C) Isopropyl alcohol treated (70%, 25 min.) L. jensenii 115-3-CHN. Please note that GFP and AlexaFluor use same laser and filter settings on the flow cytometer used in this assay