iglC Gene Research Articles

First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes.

Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faeciumNCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold;P= 0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold;P= 0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold;P= 0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold;P= 0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anti correlated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding of E. faeciumon the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action of E. faeciumon immune cell regulation in the small intestine.

Immunogenicity and Reactogenicity of Francisella tularensis Strain 15/23-1 recA - the Candidate for a New Live Tularemia Vaccine

F. tularensis strain 15/23-1 recA is different from the vaccine strain F. tularensis line 15 NIIEG by lacking one from the two copies of iglC gene and recA gene has reduced its reactogenicity for experimental animals (BALB/c mice and guinea pigs): transient weight reduction is less expressed, as well as leukopenia and thrombocytopenia during the first week after the immunization, compared to vaccine strain. The concentration of gamma-interferon in mice blood serum after 7 days immunized with strain 15/23-1 recA, was 5 times less than in mice immunized with strain 15 NIIEG. The virulence of the modified strain for mice is less than for the strain 15 NIIEG by more than two orders of magnitude. According to histological studies F. tularensis strain 15/23-1 recA stimulates morphologically a more expressed immune rearrangement in the spleen and lymph nodes as compared to F. tularensis strain 15 NIIEG. The difference of strain 15/23-1 recA from vaccine is that it is not detected in the spleens of experimental animals at 21 days after immunization. The level of specific antibodies in sera of immunized mice and guinea pigs were almost identical in both variants and there was no significant difference in the amount of gamma interferon synthesized by splenocytes of mice under specific induction. Immune mice were protected against subcutaneous challenge with virulent strain of tularemia 503 (subsp. holarctica) and protective immunity formed by the strains was lasting more than 180 days.

Construction and characterization of Francisella tularensis vaccine strain with a single copy of iglC gene and lacking recA gene

Live vaccine used in Russia against tularemia is based on Francisella tularensis subsp. holarctica NIIEG vaccine strain 15. This vaccine is highly effective, but fairly unstable. Therefore, the development of stable live tularemia vaccine with minimal side effects is rather pressing. F. tularensis iglC gene is required for intracellular production of the microbe, and recA gene is crucial for homologous recombination. Using methodology for allele removal, we deleted one copy of iglC gene and both copies of recA gene in F. tularensis vaccine strain. To achieve replacement of intact F. tularensis chromosome segments with modified segments, we constructed a novel pGM5 suicide vector based on pHV33 bireplicon plasmid. Modified chromosome segments contained a F. tularensis DNA fragment without 545 bp of iglC structural gene segment (in pGMΔiglC plasmid) and lacked a 1060-bp DNA fragment containing recA structural gene segment (pGMΔrecA plasmid). The constructed 15/23-lΔrecA mutant was capable to reproduce in macrophage-like J77A.l cell line with proliferation efficiency eight to ten times higher than parent vaccine strain 15. BALB/c mouse responded to immunization by 15/23-lΔrecA strain with a slight (about 2%) weight decrease compared to infection with strain 15 (about 14%). Bacteria of 15/23-lΔrecA strain were virtually incapable of maintaining germination in BALB/c murine spleen 14 days after invasion. In turn, bacteria of strain 15 were detected in murine spleen even after 21 days. Thus, F. tularensis 15/23-lΔrecA strain, which results in a lighter organismal reaction, can be successfully used as a basis for construction of stable live tularemia vaccines with minimal side effects.

Massively parallel delivery of large cargo into mammalian cells with light pulses.

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

Construction and Investigation of the Variants of the Vaccine Strain <I>Francisella tularensis</I> Lacking <I>iglC </I>Genes. Communication 2

for BALB/c mice by an order while retaining protective properties. Strain Francisella tularensis 15 lacking two copies of the iglC gene cannot replicate in mice organism, induces weak humoral response, is fully avirulent, and has decreased protective activity. Treatment of mice with sera obtained from the animals immunized on day 49 post immunization both with the stock F. tularensis 15 strain and its variants with one or two inactivated iglC genes has provided for 100 % protection from challenge with 200 DCL of F. tularensis 15 strain. However in case of BALB/c mice exposure to virulent F. tularensis 503 and F. tularensis Schu strains (50 DCL and 25 DSL, respectively), treated with immune sera 24 hours before, registered has been only mean lifetime increase.

Construction and Investigation of the Vaccine Strain Francisella tularensis without iglC genes. Communication 1

Using site-specific mutagenesis constructed were the variants of the vaccine strain F. tularensis subsp. holarctica 15 with the deleted iglC genes. Identified was the fact that genome of the strain 15 contained two copies of iglC gene. Deletion of one of them as well as both had little effect on the cultural-morphological and growth properties of the microbe. At the same time F. tularensis 15 lacking one copy of the iglC gene propagated in mice macrophages several times slower, than the original strain. Inactivation of both of the copies in the chromosome leaded to the emergence of a variant incapable of intracellular reproduction. This capacity in F. tularensis 15/23-2 with two inactivated iglC gene copies was partially recovered after integration of a complementing plasmid. Therewith the data mentioned above testifies to the significance of iglC gene for the process of reproduction in macrophages.

Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions

BackgroundReverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species.ResultsRelative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene.ConclusionAlthough the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.

Francisella noatunensis subsp. orientalis pathogenesis analyzed by experimental immersion challenge in Nile tilapia, Oreochromis niloticus (L.)

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent warmwater fish pathogen and the causative agent of francisellosis in tilapia (Oreochromis sp). To study the pathogenesis of this bacterium, tilapia fingerlings were experimentally infected by immersion challenge with wild type (WT) Fno and the distribution of bacteria to multiple organs, as well as associated lesion development, investigated after 3, 24, 48, 96, and 192h by real-time PCR and histopathological examination. Surface mucus collected 3h post-infection contained the highest number of Fno genome equivalents (GE). After 96h, marked increases of WT Fno GE were detected in spleen, anterior kidney, posterior kidney, gill, heart, liver, brain, gonad, and the gastrointestinal tract. Increases in bacterial GE also corresponded to the appearance, size and number of granulomas typical of francisellosis, predominantly in the spleen and anterior and posterior kidney segments. A simultaneous comparison was also made in tilapia challenged with an attenuated Fno strain containing a mutation in the intracellular growth locus C (iglC) gene, essential to intracellular survival. Compared to the WT, the mutant iglC strain was present in most tissues in similar numbers prior to 48h post-challenge. While the mutant did not replicate significantly or produce lesions in any tissue, it persisted for up to 192h. These findings provide insight into the pathophysiology of francisellosis in tilapia, which may also prove useful as a model for the study of mammalian tularemia, and advance our understanding of the utility of the ΔiglC mutant as a potential vaccine candidate.

Molecular and immunohistochemical diagnosis of Francisella noatunensis subsp. orientalis from formalin-fixed, paraffin-embedded tissues

Members of the genus Francisella (viz., F. noatunensis subsp. orientalis [Fno] and F. noatunensis subsp. noatunensis) have been described as causative agents of chronic granulomatous and pyogranulomatous lesions in wild and cultured fish species. In the present study, 68 archived formalin-fixed, paraffin-embedded (FFPE) tissues from several fish species, collected at different geographical locations from 2000 to 2011, were analyzed using a real-time polymerase chain reaction assay for the detection of the Fno intracellular growth loci C (iglC) gene and by immunohistochemistry for the demonstration of Fno antigens. The results revealed a high correlation between these 2 diagnostic techniques validating their use for the diagnosis of Fno infection in archived FFPE tissues and confirming the presence of Fno in fish species from the Cari y years of the present century.

Francisella asiatica as the causative agent of piscine francisellosis in cultured tilapia (Oreochromis sp.) in the United States

Francisella asiatica is a Gram-negative, pleomorphic, facultative intracellular, bacterial pathogen that causes acute to chronic disease in a wide variety of warm-water cultured and wild fish species. Outbreaks of francisellosis in warm water fish have been documented in Taiwan, Japan, United Kingdom, Hawaii, and Latin America (including Costa Rica) but the organism has only been reported from the United States on one occasion from hybrid striped bass in California. In 2010, the bacterium was detected from diseased tilapia by culture on cystine heart agar supplemented with hemoglobin and by utilizing an F. asiatica-specific real-time polymerase chain reaction assay. The tilapia (Oreochromis niloticus) were cultured in an indoor, closed, recirculating aquaculture facility in the Midwest of the United States. The identity of isolates recovered from diseased fish was confirmed as F. asiatica by amplification and sequence comparison of the 16S ribosomal RNA and intracellular growth locus C (iglC) gene. Gross and microscopic examination of affected tissues revealed the presence of marked anterior renomegaly and splenomegaly with severe granulomatous disease.

Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis

Members of the genus Francisella are small Gram-negative facultative intracellular bacteria that cause francisellosis in a wide variety of fish species worldwide. F. noatunensis subsp. orientalis has been recently described as a warm-water pathogen of tilapia Oreochromis spp. In this study, a quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay was developed to rapidly and accurately detect and quantify F. noatunensis subsp. orientalis from fish tissue. The target region of the assay was the F. tularensis iglC gene homologue previously found in F. noatunensis subsp. orientalis. Probe specificity was confirmed by the lack of signal and cross-reactivity with 12 common fish pathogens, 2 subspecies of F. tularensis, F. noatunensis subsp. noatunensis, and tilapia tissue. The range of linearity was determined to be 50 fg to 1.4 mg, and the lower limit of detection was 50 fg of DNA (equivalent to approximately 25 genome equivalents) per reaction. A similar sensitivity was observed with DNA extracted from a mixture of F. noatunensis subsp. orientalis and fish tissue. The assay was also able to detect and quantify F. noatunensis subsp. orientalis from the spleens of experimentally infected tilapia. No signal was observed in the control groups. In conclusion, we have developed a highly sensitive and specific assay that can be used for the specific identification and quantification of F. noatunensis subsp. orientalis.

Factors affecting the escape of Francisella tularensis from the phagolysosome.

The highly virulent bacterium Francisella tularensis is well adapted to the intracellular habitat but the mechanisms behind its intracellular survival have been elusive. Recently, it was shown that the bacterium is capable of escaping from the phagosome of human and mouse monocytic cells. Here it is shown that this escape is affected by gamma interferon (IFN-gamma) treatment of mouse peritoneal exudate cells since in treated cells the proportion that escaped was significantly lower (80%) than in untreated cells (97%) as determined by transmission electron microscopy. By contrast, < 1% of mutant bacteria lacking expression of a 23 kDa protein denoted IglC were able to escape from the phagosome. Infection with the DeltaiglC strain complemented with the iglC gene resulted in 60% of the bacteria escaping from the phagosome. Whereas IFN-gamma treatment conferred a static effect on intracellular wild-type bacteria, the treatment had a bactericidal effect on the DeltaiglC strain. The results show that the activation status of infected cells affects the escape of F. tularensis from the phagosome. An even more profound effect on this escape is related to expression of IglC by F. tularensis. Its absence rendered the mutant bacteria incapable of escaping from the phagosome and of multiplying intracellularly.

Binding of free immunoglobulin light chains to VpreB3 inhibits their maturation and secretion in chicken B cells.

The VpreB3 gene product was first characterized as an immunoglobulin (Ig) mu heavy chain-binding protein in mouse precursor B (pre-B) cells. Although its function is unknown, it has been proposed to participate in the assembly and transport of the pre-B cell receptor. We have identified a VpreB3 orthologous gene in chicken that is located close to the immunoglobulin light chain (LC) gene cluster and specifically expressed in the bursa of Fabricius. By overexpressing VpreB3 in the DT40 IgM(+) immature chicken B cell line, we have characterized VpreB3 as an endoplasmic reticulum-resident glycoprotein that binds preferentially to free IgLC. However, binding to IgHC is observed in IgLC-deficient DT40 cells. Interaction of VpreB3 with free IgLC is partly covalent and induces retention of free IgLC in the endoplasmic reticulum, preventing their secretion without affecting IgM surface expression. Our results demonstrate that this evolutionarily conserved molecule may play a role in the regulation of the maturation and secretion of free IgLC in B cells. We discuss possible implications in the regulation of the immune response.

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-Δ iglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted Δ iglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated Δ iglC1+2, was obtained. The Δ iglC1+2 strain, in contrast to Δ iglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.

Structure and diversity of Mexican axolotl lambda light chains

We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl.

Mapping of the human lambda immunoglobulin variable gene subgroup 1.

We describe the cloning and mapping of 20 putative members of the IGLV subgroup 1. These gene segments are contained on 26 phage clones which fall into 7 contigs plus one solitary phage. This represents approximately 240 kilobases (kb) of cloned DNA. Like IGLC gene segments, the IGLV gene segments were found to be oriented proximal to distal on the chromosome, indicating IGL somatic rearrangement is by deletion. The gene segments were placed on a long-range map of the IGL locus, which covers at least 800 kb. Clones were further ordered by pulsed field gel electrophoresis analysis of B-cell lines known to produce IGL-containing immunoglobulins. DNA deletions ranged from 120 to 570 kb.