Cloning of an outer membrane protein of Neisseria meningitidis in Escherichia coli

A gene bank of chromosomal DNA of Neisseria meningitidis group B was constructed in phage lambda EMBL3, and screened by rabbit polyclonal antibodies to major outer membrane (OM) proteins of the meningococcus. Several clones expressing a 28 kDa protein were found. The gene coding for the 28 kDa protein was subcloned into plasmid pUC18 in Escherichia coli. The protein was expressed in E. coli and located in the OM. Rabbit antibodies were raised to the 28 kDa protein purified from E. coli and used to localize the protein in the meningococcus. The antiserum recognized a minor protein of similar electrophoretic mobility in the outer membrane complex (OMC) of the meningococcus. The 28 kDa protein was found to be common to different Neisseria species; it was expressed by both pathogenic and nonpathogenic Neisseria.     

Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.     

TLR2 and TLR4 polymorphisms in familial Mediterranean fever

It has been suggested that MEVF mutations offer advantage against infections, including tuberculosis. Bearing in mind the central role of TLR-2 and TLR-4 in the recognition of pathogens, we conducted this study to examine whether the TLR2-R753Q, TLR4-D299G, TLR4-T399I common polymorphisms are associated with susceptibility to familial Mediterranean fever (FMF) or affect the course of the disease. A cohort of 169 FMF patients and 245 healthy bone marrow donors were enrolled in the study. FMF patients appeared with a significantly lower frequency of the TLR4-D299G mutated allele (3.2% vs 6.9%, p = 0.032). No association was observed with the other analyzed polymorphisms. Moreover, we found no association between polymorphisms and the frequency of attacks or the development of amyloidosis. Our results may reinforce the hypothesis that FMF patients display a better defense against pathogens, providing an additional mechanism and suggesting a positive selection advantage in the area of the Mediterranean basin.     

In vivo detection of lamellocytes in Drosophila melanogaster

Drosophila has recently become a powerful model organism for studies of innate immunity. The cellular elements of innate immunity in Drosophila, the hemocytes, have been characterized by morphological criteria, molecular markers, and cell-type-specific immunological markers. Here we suggest that an MiET1 GFP-reporter element insertion in the untranslated region of a gene (l1-atilla) – expressed in a subset of hemocytes, the lamellocytes – allows in vivo investigations of lamellocyte differentiation and facilitates genetic screens.     

Peptide epitopes of the Taenia solium antigen Ts8B2 are immunodominant in human and porcine cysticercosis

Ts8B2 is a gene which encodes for a member of the Taenia solium metacestode 8 kDa antigen family. Since the Ts8B2-GST recombinant protein compares very favourably with other diagnostic antigens, and in order to study the antigenic nature and structure of this molecule, the Ts8B2 was expressed in prokaryotic and eukaryotic systems. The diagnostic potential of the recombinant Ts8B2 proteins was evaluated by enzyme-linked immunosorbent assays (ELISA) using a collection of serum and cerebrospinal fluid (CSF) samples from patients with clinically defined neurocysticercosis (NCC), and also sera from T. solium infected pigs. Despite the predicted glycosylation of the Ts8B2-Bac recombinant protein, there was very little difference in assay sensitivity/specificity when the Ts8B2 reagent was expressed in either prokaryotic or eukaryotic systems, suggesting that peptidic Ts8B2 epitopes are immunodominant in porcine cysticercosis and human neurocysticercosis. Conveniently, production of recombinant Ts8B2 in Escherichia coli is economical and facile, making it a feasible and practical choice as a diagnostic reagent for use in endemic areas. The Ts8B2 ELISA is particularly useful for the diagnosis of active as opposed to inactive cases of NCC and conduct of the assay is also facilitated by the fact that assay sensitivity is significantly greater when serum as opposed to CSF samples are employed.     

RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi

In this study, we used real-time PCR to simultaneously monitor the responses of 12 key genes of the shrimp innate immune system in Litopenaeus vannamei after challenge with Vibrio harveyi. In the proPO activating system, we found that proPO was up-regulated (3.3× control at 36 hpi). The hemolymph clotting genes transglutaminase (TGase) and clotting protein were also up-regulated, as were 5 genes in the antimicrobial peptide system (ALF, Crustin, Lyz, PEN2 and PEN4), with only PEN3 showing no significant changes. In the antioxidant defense system, SOD was slightly elevated while GPx was substantially down-regulated. In the pattern recognition receptor system, at 24 hpi, the Toll gene (LvToll) showed the highest relative increase in expression level of all the investigated genes (15× greater than the sterile seawater control). In the second part of this study, when LvToll was knocked down by RNAi silencing, there was no effect on either survival rates or bacterial number in unchallenged shrimp. There was also no difference in mortality rates between control shrimp and LvToll-silenced shrimp when these two groups were challenged with a viral pathogen (white spot syndrome virus; WSSV). However, when LvToll-silenced shrimp were challenged by V. harveyi, there was a significant increase in mortality and bacterial CFU counts. We note that the increase in bacterial CFU count occurred even though treatment with EGFP dsRNA had the opposite effect of reducing the CFU counts. We conclude that LvToll is an important factor in the shrimp innate immune response to acute V. harveyi infection, but not to WSSV.     

Identification of Rhodococcus, Gordona and Dietzia species using carbon source utilization tests (“Biotype-100” strips)

The “Biotype-100” identification system (BioMérieux, La Balme-les-Grottes, France) based on carbon source utilization was evaluated for its ability to discriminate among 10 species of Rhodococcus, 7 species of Gordona and one species of Dietzia. The type strains of three species of Tsukamurella and 8 species of Nocardia were also included in the study. Results were compared with chemotaxonomic and conventional data. Carbon source utilization was shown to be reliable, rapid and easy to use when compared with standard identification methods. The 29 species tested were unambiguously separated by carbon source utilization tests. Rhodococcus equi was found to be heterogenous.Les galeries “Biotype-100” (BioMérieux) ont été utilisées pour différencier 10 espèces du genre Rhodococcus, 7 espèces du genre Gordona et 1 espèce du genre Dietzia entre elles. Les souchestypes de 3 espèces du genre Tsukamurella et 8 espèces du genre Nocardia ont été incluses dans cette étude. Les résultats ont été comparés avec ceux des études chimiotaxonomiques et ceux obtenus avec les galeries d'identification classiques. Les galeries Biotype-100 sont sûres, rapides et faciles à utiliser par rapport aux galeries classiques. Les 29 espèces étudiées ont été identifiées sans aucune difficulté. L'espèce Rhodococcus equi s'est révélée hétérogène.     

Characterization of a 30-kDa Borrelia burgdorferi substrate-binding protein homologue

A Borrelia burgdorferi chromosomal gene encodes a 30-kDa antigen (P30) that has considerable homology with periplasmic substrate-binding proteins of Gram-negative bacteria, and is recognized by antibodies in sera from a subset of patients with Lyme disease and from B. burgdorferi-infected mice. The p30 gene is 801 nucleotides in length and P30 contains 267 amino acids, with predicted molecular mass of 30 kDa. The P30 amino acid region 36–258 has homology to conserved domains of the oligopeptide permease A of Gram-negative bacteria. Immunofluorescence studies using murine anti-P30 serum suggest that P30 is on the outer surface of B. burgdorferi. P30 expression could be detected in representatives of all 3 subspecies of B. burgdorferi sensu lato, but not in all of the tested strains. Antibodies to P30 were detected in sera of 18 out of 82 patients (22%) with Lyme disease, including individuals with early- or late-stage infection. Although antibodies to P30 are present in the sera of C3H/HeN mice infected with B. burgdorferi for at least 90 days, immunization with recombinant P30 does not protect mice from infection. We conclude that P30 is a putative substrate-binding protein of B. burgdorferi and is immunologically recognized in human and murine Lyme borreliosis.     

First molecular evidence of Tula hantavirus in Microtus voles in Slovenia

Different Microtus species, present in a worldwide range habitat populating North America, Europe, Asia, and few other species have been recognized previously as a hantavirus reservoir. Tula hantavirus was first reported in Microtus arvalis and Microtus rossiaemeridionalis from Central Russia and later discovered in several European countries. Using molecular techniques we have demonstrated the presence of Tula hantavirus in three different Microtus species in Slovenia. Phylogenetic analyses of partial S segment placed Slovenian strains in the same genetic lineage as Austrian and Croatian strains.     

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin–haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.     

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.     

The A312L 5′-UTR of Chlorella virus PBCV-1 is a translational enhancer in Arabidopsis thaliana

PBCV-1 (Paramecium bursaria Chlorella virus) is a large double stranded DNA virus that replicates in certain eukaryotic chlorella like green algae. The PBCV-1 A312L gene encodes a 33-kDa protein whose function currently is unknown. The 5′-UTR of the A312L mRNA is 153 nucleotides, longer than the 5′-UTR in any other PBCV-1 gene. The sequence 5′-AAAC was repeated 17 times within 156 bp 5′ to the A312L gene start codon and this sequence was repeated 13 times continuously in the 5′-UTR of the mRNA. Recombinant genes were constructed in vector pBI121 that contained the A312L 5′-UTR, in both the forward and inverse-complement orientations, fused to the GUS gene under the control of the CaMV 35S promoter. These constructs were introduced into Arabidopsis thaliana and the results indicated that the A312L 5′-UTR functions as a translational enhancer only in the forward orientation. Overall, the ratio of GUS enzyme activity to GUS mRNA was 15-fold higher in constructs derived from the A312L 5′-UTR in the forward orientation as compared to constructs containing the 5′-UTR in the inverse-complement orientation or those lacking the A312L 5′-UTR.     

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58–68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.