High-Throughput Screening for the Prevalence of Neutralizing Antibodies against Human Adenovirus Serotype 5.

Adenoviral vectors based on the human adenovirus species C serotype 5 (HAdV-C5) are commonly used for vector-based gene therapies and vaccines. In the preclinical stages of development, their safety and efficacy are often validated in suitable animal models. However, pre-existing neutralizing antibodies may severely influence study outcomes. Here, we generated a new HAdV-C5-based reporter vector and established a high-throughput screening assay for the multivalent detection of HAdV-C5-neutralizing antibodies in serum. We screened the sera of rhesus macaques at different primate centers, and of rabbits, horses, cats, and dogs, showing that HAdV-C5-neutralizing antibodies can be found in all species, albeit at different frequencies. Our results emphasize the need to prescreen model animals in HAdV-C5-based studies.

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae.

IMPORTANCE: This study highlights diversity in iron acquisition and regulation in bacteria. The mechanisms of iron acquisition and its regulation in Teredinibacter turnerae, as well as its connection to cellulose utilization, a hallmark phenotype of T. turnerae, expand the paradigm of bacterial iron acquisition. Two of the four TonB genes identified in T. turnerae exhibit functional redundancy and play a crucial role in siderophore-mediated iron transport. Unlike typical TonB genes in bacteria, none of the TonB genes in T. turnerae are clearly iron regulated. This unusual regulation could be explained by another important finding in this study, namely, that the two TonB genes involved in iron transport are also essential for cellulose utilization as a carbon source, leading to the expression of TonB genes even under iron-rich conditions.

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae.

Teredinibacter turnerae is an intracellular bacterial symbiont that resides in the gills of shipworms, wood-eating bivalve mollusks. This bacterium produces a catechol siderophore, turnerbactin, required for the survival of this bacterium under iron limiting conditions. The turnerbactin biosynthetic genes are contained in one of the secondary metabolite clusters conserved among T. turnerae strains. However, Fe(III)-turnerbactin uptake mechanisms are largely unknown. Here, we show that the first gene of the cluster, fttA a homologue of Fe(III)-siderophore TonB-dependent outer membrane receptor (TBDR) genes is indispensable for iron uptake via the endogenous siderophore, turnerbactin, as well as by an exogenous siderophore, amphi-enterobactin, ubiquitously produced by marine vibrios. Furthermore, three TonB clusters containing four tonB genes were identified, and two of these genes, tonB1b and tonB2, functioned not only for iron transport but also for carbohydrate utilization when cellulose was a sole carbon source. Gene expression analysis revealed that none of the tonB genes and other genes in those clusters were clearly regulated by iron concentration while turnerbactin biosynthesis and uptake genes were up-regulated under iron limiting conditions, highlighting the importance of tonB genes even in iron rich conditions, possibly for utilization of carbohydrates derived from cellulose.

Correction to: Amphi­enterobactin commonly produced among Vibrio campbellii and Vibrio harveyi strains can be taken up by a novel outer membrane protein FapA that also can transport canonical Fe(III)­enterobactin.

In the original publication, third author's name was incorrectly published as Aneta L. Jelowicki.

Amphi-enterobactin commonly produced among Vibrio campbellii and Vibrio harveyi strains can be taken up by a novel outer membrane protein FapA that also can transport canonical Fe(III)-enterobactin.

Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism for quorum sensing study and produces the siderophores anguibactin and amphi-enterobactin. This study examined the mechanisms and specificity of siderophore uptake in V. campbellii and V. harveyi, and surveyed the diversity of siderophore production in V. campbellii and V. harveyi strains. The amphi-enterobactin gene cluster of BAA-1116 harbors a gene, named fapA, that is a homologue of genes encoding Fe(III)-siderophore-specific outer membrane receptors. Another strain, V. campbellii HY01, a strain pathogenic to shrimp, also carries this cluster including fapA. Our siderophore bioassay results using HY01-derived indicator strains show that the FapA protein localized in the outer membrane fraction of V. campbellii HY01 is essential for the uptake of Fe(III)-amphi-enterobactin as well as exogenous siderophores, including enterobactin from E. coli, but not vanchrobactin from V. anguillarum RV22 while Fe(III)-amphi-enterobactin can be utilized by V. anguillarum. Electrospray ionization mass spectrometry as well as bioassay revealed that various V. campbellii and V. harveyi strains produce a suite of amphi-enterobactins with various fatty acid appendages, including several novel amphi-enterobactins, and these amphi-enterobactins can be taken up by V. campbellii HY01 via FapA, indicating that amphi-enterobactin production is a common phenotype among V. campbellii and V. harveyi, whereas our previous work, confirmed herein, showed that anguibactin is only produced by V. campbellii strains. These results along with the additional finding that a 2,3-dihydroxybenzoic acid biosynthesis gene, aebA, located in the amphi-enterobactin gene cluster, is essential for both anguibactin and amphi-enterobactin biosynthesis, suggest the possibility that amphi-enterobactin is a native siderophore of V. campbellii and V. harveyi, while the anguibactin system has been acquired by V. campbellii during evolution.

Fatty acid hydrolysis of acyl marinobactin siderophores by Marinobacter acylases.

The marine bacteria Marinobacter sp. DS40M6 and Marinobacter nanhaiticus D15-8W produce a suite of acyl peptidic marinobactin siderophores to acquire iron under iron-limiting conditions. During late-log phase growth, the marinobactins are hydrolyzed to form the marinobactin headgroup with release of the corresponding fatty acid tail. The bntA gene, a homologue of the Pseudomonas aeruginosa pyoverdine acylase gene, pvdQ, was identified from Marinobacter sp. DS40M6. A bntA knockout mutant of Marinobacter sp. DS40M6 produced the suite of acyl marinobactins A-E, without the usual formation of the marinobactin headgroup. Another marinobactin-producing species, M. nanhaiticus D15-8W, is predicted to have two pvdQ homologues, mhtA and mhtB. MhtA and MhtB have 67% identical amino acid sequences. MhtA catalyzes hydrolysis of the apo-marinobactin siderophores as well as the quorum sensing signaling molecule, dodecanoyl-homoserine lactone. In contrast to hydrolysis of the suite of apo-marinobactins by MhtA, hydrolysis of the iron(III)-bound marinobactins was not observed.

Biosynthesis of amphi-enterobactin siderophores by Vibrio harveyi BAA-1116: identification of a bifunctional nonribosomal peptide synthetase condensation domain.

The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.

Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution.

Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.

Two ABC transporter systems participate in siderophore transport in the marine pathogen Vibrio anguillarum 775 (pJM1).

ORF40 (named fatE) in the Vibrio anguillarum pJM1 plasmid-encoding anguibactin iron transport systems is a homolog of ATPase genes involved in ferric-siderophore transport. Mutation of fatE did not affect ferric-anguibactin transport, indicating that there must be other ATPase gene(s) in addition to fatE. By searching the genomic sequence of V. anguillarum 775(pJM1), we identified a homolog of fatE named fvtE on chromosome 2. It is of interest that in this locus, we also identified homologs of fatB, fatC, and fatD that we named fvtB, fvtC and fvtD, respectively. The fvtE mutant still showed ferric-anguibactin transport, while the double fatE and fvtE mutation completely abolished the ferric-anguibactin transport indicating that fatE and fvtE are functional ATPase homologs for ferric-anguibactin transport. Furthermore, we demonstrate that fvtB, fvtC, fvtD, and fvtE are essential for ferric-vanchrobactin and ferric-enterobactin transport.

The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum?

Many Vibrio anguillarum serotype O1 strains carry 65-kb pJM1-type plasmids harboring genes involved in siderophore anguibactin biosynthesis and transport. The anguibactin system is an essential factor for V. anguillarum to survive under iron-limiting conditions, and as a consequence, it is a very important virulence factor of this bacterium. Our comparative analysis of genomic data identified a cluster harboring homologs of anguibactin biosynthesis and transport genes in the chromosome of Vibrio harveyi. We have purified the putative anguibactin siderophore and demonstrated that it is indeed anguibactin by mass spectrometry and specific bioassays. Furthermore, we characterized two genes, angR and fatA, in this chromosome cluster that, respectively, participate in anguibactin biosynthesis and transport as determined by mutagenesis analysis. Furthermore, we found that the V. harveyi FatA protein is located in the outer membrane fractions as previously demonstrated in V. anguillarum. Based on our data, we propose that the anguibactin biosynthesis and transport cluster in the V. anguillarum pJM1 plasmid have likely evolved from the chromosome cluster of V. harveyi or vice versa.

Genome sequence of the marine bacterium Vibrio campbellii DS40M4, isolated from open ocean water.

Vibrio sp. strain DS40M4 is a marine bacterium that was isolated from open ocean water. In this work, using genomic taxonomy, we were able to classify this bacterium as V. campbellii. Our genomic analysis revealed that V. campbellii DS40M4 harbors genes related to iron transport, virulence, and environmental fitness, such as those encoding anguibactin and vanchrobactin biosynthesis proteins, type II, III, IV, and VI secretion systems, and proteorhodopsin.

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum.

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.

Identification and characterization of a novel outer membrane protein receptor FetA for ferric enterobactin transport in Vibrio anguillarum 775 (pJM1).

In this work we demonstrate the existence in Vibrio anguillarum 775 (pJM1) of two chromosomal genes encoding outer membrane proteins that operate in the transport of ferric enterobactin. One of them is a novel receptor that we named FetA and the other is the already characterized FvtA that functions in the uptake of iron complexes of both enterobactin and vanchrobactin. Ferric enterobactin transport proficiency was resumed in double mutants for these two genes when they were complemented with either fetA or fvtA, whereas only the cloned fvtA could complement for ferric vanchrobactin transport. Quantitative RT-PCR assays demonstrated that transcription of the fetA gene is regulated by FetR, that is encoded upstream and in reverse orientation from fetA. This gene as well as fetA, are up-regulated in iron limiting condition in a Fur-dependent manner. The two divergent promoters are located in the intergenic region between fetR and fetA that has a putative Fur binding site and an IrgB binding site in the overlapping promoters of fetR and fetA. FetA and FetR show high homology to V. cholerae IrgA and IrgB respectively and the intergenic regions fetA-fetR and irgA-irgB are also highly related suggesting a vertical transmission of the fetA-fetR cluster from V. cholerae to V. anguillarum.

Genetic Determinants of Virulence in the Marine Fish Pathogen Vibrio anguillarum.

One of the most studied fish pathogens is Vibrio anguillarum. Development of the genetics and biochemistry of the mechanisms of virulence in this fish pathogen together with clinical and ecologic studies has permitted the intensive development of microbiology in fish diseases. It is the intention of this review to compile the exhaustive knowledge accumulated on this bacterium and its interaction with the host fish by reporting a complete analysis of the V. anguillarum virulence factors and the genetics of their complexity.

Complete genome sequence of the marine fish pathogen Vibrio anguillarum harboring the pJM1 virulence plasmid and genomic comparison with other virulent strains of V. anguillarum and V. ordalii.

We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2ß (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host "addiction" genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.

The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980)--a taxonomic opinion.

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437(T) and ATCC 68554 (=strain 775) was Vibrio ordalii LMG 13544(T), with more than 99.5% 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544(T), with 95% AAI, 98% MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77% similarity in AAI, 85% in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67% DNA-DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges-Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

Role of the pJM1 plasmid-encoded transport proteins FatB, C and D in ferric anguibactin uptake in the fish pathogen Vibrio anguillarum.

Vibrio anguillarum serotype O1 is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal haemorrhagic septicemia in marine and fresh water fish due to the action of the anguibactin iron uptake system encoded by the virulence plasmid pJM1. This plasmid harbours the genes for the biosynthesis of the siderophore anguibactin and the ferric anguibactin transport proteins FatD, C, B and A encoded in the iron transport operon. The FatA protein is the outer membrane receptor for the ferric siderophore complex and the FatB lipoprotein provides the periplasmic domain for its internalization, whereas the FatC and D proteins are located in the cytoplasmic membrane and might play a role as part of the ABC transporter for internalization of the ferric siderophore. In this work we demonstrate the essential role of these two inner membrane proteins in ferric anguibactin transport and that the lipo-protein nature of FatB is not necessary for ferric anguibactin transport.

HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6.

In Vibrio vulnificus, HlyU upregulates the expression of the large RTX toxin gene. In this work we identified the binding site of HlyU to -417 to -376 bp of the rtxA1 operon transcription start site. lacZ fusions for a series of progressive deletions from the rtxA1 operon promoter showed that transcriptional activity increased independently of HlyU when its binding site was absent. Thus HlyU must regulate the rtxA1 operon expression by antagonizing a negative regulator. Concomitantly we found that an hns mutant resulted in an increase in the expression of the rtxA1 operon genes. Multiple copies of HlyU can increase the promoter activity only in the presence of H-NS underscoring the hypothesis that HlyU must alleviate the repression by this protein. H-NS binds to a region that extends upstream and downstream of the rtxA1 operon promoter. In the upstream region it binds to five AT-rich sites of which two overlap the HlyU binding site. Competitive footprinting and gel shift data demonstrate HlyU's higher affinity as compared with H-NS resulting in the de-repression and a corresponding increased expression of the rtxA1 operon.

Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum.

Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa predicted protein, AngN. In this work we show that AngN is essential for anguibactin biosynthesis and possesses two domains with homology to cyclization (Cy) domains of NRPSs. Substitution by alanine of the aspartic acid residues within a conserved motif of either Cy1 or Cy2 domain demonstrated the importance of these two domains in AngN function during siderophore biosynthesis. Site-directed mutations in both domains (D133A/D575A and D138A/D580A) resulted in anguibactin-deficient phenotypes while mutations in each domain did not abolish siderophore production but caused a reduction in the amounts produced. The mutations D133A/D575A and D138A/D580A also resulted as expected in a dramatic attenuation of the virulence of V. anguillarum 775 highlighting the importance of this gene for the biosynthesis of anguibactin within the vertebrate host. Regulation of the angN gene follows the patterns observed at the iron transport-biosynthesis promoter with angN transcription repressed in the presence of iron and enhanced by AngR and trans-acting factor (TAF) under iron limitation.

Global gene expression as a function of the iron status of the bacterial cell: influence of differentially expressed genes in the virulence of the human pathogen Vibrio vulnificus.

Vibrio vulnificus multiplies rapidly in host tissues under iron-overloaded conditions. To understand the effects of iron in the physiology of this pathogen, we performed a genome-wide transcriptional analysis of V. vulnificus growing at three different iron concentrations, i.e., iron-limiting [Trypticase soy broth with 1.5% NaCl (TSBS) plus ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA)], low-iron (1 microg Fe/ml; TSBS), and iron-rich (38 microg Fe/ml; TSBS plus ferric ammonium citrate) concentrations. A few genes were upregulated under the last two conditions, while several genes were expressed differentially under only one of them. A gene upregulated under both conditions encodes the outer membrane porin, OmpH, while others are related to the biosynthesis of amino sugars. An ompH mutant showed sensitivity to sodium dodecyl sulfate (SDS) and polymyxin B and also had a reduced competitive index compared with the wild type in the iron-overloaded mice. Under iron-limiting conditions, two of the TonB systems involved in vulnibactin transport were induced. These genes were essential for virulence in the iron-overloaded mice inoculated subcutaneously, underscoring the importance of active iron transport in infection, even under the high-iron conditions of this animal model. Furthermore, we demonstrated that a RyhB homologue is also essential for virulence in the iron-overloaded mouse. This novel information on the role of genes induced under iron limitation in the iron-overloaded mouse model and the finding of new genes with putative roles in virulence that are expressed only under iron-rich conditions shed light on the many strategies used by this pathogen to multiply rapidly in the susceptible host.