A Zoonotic Adenoviral Human Pathogen Emerged through Genomic Recombination among Human and Nonhuman Simian Hosts.

Genomics analysis of a historically intriguing and predicted emergent human adenovirus (HAdV) pathogen, which caused pneumonia and death, provides insight into a novel molecular evolution pathway involving "ping-pong" zoonosis and anthroponosis. The genome of this promiscuous pathogen is embedded with evidence of unprecedented multiple, multidirectional, stable, and reciprocal cross-species infections of hosts from three species (human, chimpanzee, and bonobo). This recombinant genome, typed as HAdV-B76, is identical to two recently reported simian AdV (SAdV) genomes isolated from chimpanzees and bonobos. Additionally, the presence of a critical adenoviral replication element found in HAdV genomes, in addition to genes that are highly similar to counterparts in other HAdVs, reinforces its potential as a human pathogen. Reservoirs in nonhuman hosts may explain periods of apparent absence and then reemergence of human adenoviral pathogens, as well as present pathways for the genesis of those thought to be newly emergent. The nature of the HAdV-D76 genome has implications for the use of SAdVs as gene delivery vectors in human gene therapy and vaccines, selected to avoid preexisting and potentially fatal host immune responses to HAdV.IMPORTANCE An emergent adenoviral human pathogen, HAdV-B76, associated with a fatality in 1965, shows a remarkable degree of genome identity with two recently isolated simian adenoviruses that contain cross-species genome recombination events from three hosts: human, chimpanzee, and bonobo. Zoonosis (nonhuman-to-human transmission) and anthroponosis (human to nonhuman transmission) may play significant roles in the emergence of human adenoviral pathogens.

Genomics-based re-examination of the taxonomy and phylogeny of human and simian Mastadenoviruses: an evolving whole genomes approach, revealing putative zoonosis, anthroponosis, and amphizoonosis.

With the advent of high-resolution and cost-effective genomics and bioinformatics tools and methods contributing to a large database of both human (HAdV) and simian (SAdV) adenoviruses, a genomics-based re-evaluation of their taxonomy is warranted. Interest in these particular adenoviruses is growing in part due to the applications of both in gene transfer protocols, including gene therapy and vaccines, as well in oncolytic protocols. In particular, the re-evaluation of SAdVs as appropriate vectors in humans is important as zoonosis precludes the assumption that human immune system may be naïve to these vectors. Additionally, as important pathogens, adenoviruses are a model organism system for understanding viral pathogen emergence through zoonosis and anthroponosis, particularly among the primate species, along with recombination, host adaptation, and selection, as evidenced by one long-standing human respiratory pathogen HAdV-4 and a recent re-evaluation of another, HAdV-76. The latter reflects the insights on amphizoonosis, defined as infections in both directions among host species including "other than human", that are possible with the growing database of nonhuman adenovirus genomes. HAdV-76 is a recombinant that has been isolated from human, chimpanzee, and bonobo hosts. On-going and potential impacts of adenoviruses on public health and translational medicine drive this evaluation of 174 whole genome sequences from HAdVs and SAdVs archived in GenBank. The conclusion is that rather than separate HAdV and SAdV phylogenetic lineages, a single, intertwined tree is observed with all HAdVs and SAdVs forming mixed clades. Therefore, a single designation of "primate adenovirus" (PrAdV) superseding either HAdV and SAdV is proposed, or alternatively, keeping HAdV for human adenovirus but expanding the SAdV nomenclature officially to include host species identification as in ChAdV for chimpanzee adenovirus, GoAdV for gorilla adenovirus, BoAdV for bonobo adenovirus, and ad libitum.

Iron-regulated small RNA expression as Neisseria gonorrhoeae FA 1090 transitions into stationary phase growth.

BACKGROUND: For most pathogens, iron (Fe) homeostasis is crucial for maintenance within the host and the ability to cause disease. The primary transcriptional regulator that controls intracellular Fe levels is the Fur (ferric uptake regulator) protein, which exerts its action on transcription by binding to a promoter-proximal sequence termed the Fur box. Fur-regulated transcriptional responses are often fine-tuned at the post-transcriptional level through the action of small regulatory RNAs (sRNAs). Consequently, identifying sRNAs contributing to the control of Fe homeostasis is important for understanding the Fur-controlled bacterial Fe-response network. RESULTS: In this study, we sequenced size-selected directional libraries representing sRNA samples from Neisseria gonorrhoeae strain FA 1090, and examined the Fe- and temporal regulation of these sRNAs. RNA-seq data for all time points identified a pool of at least 340 potential sRNAs. Differential analysis demonstrated that expression appeared to be regulated by Fe availability for at least fifteen of these sRNAs. Fourteen sRNAs were induced in high Fe conditions, consisting of both cis and trans sRNAs, some of which are predicted to control expression of a known virulence factor, and one SAM riboswitch. An additional putative cis-acting sRNA was repressed by Fe availability. In the pathogenic Neisseria species, one sRNA that contributes to Fe-regulated post-transcriptional control is the Fur-repressible sRNA NrrF. The expression of five Fe-induced sRNAs appeared to be at least partially controlled by NrrF, while the remainder was expressed independently of NrrF. The expression of the 14 Fe-induced sRNAs also exhibited temporal control, as their expression levels increased dramatically as the bacteria entered stationary phase. CONCLUSIONS: Here we report the temporal expression of Fe-regulated sRNAs in N. gonorrhoeae FA 1090 with several appearing to be controlled by the Fe-repressible sRNA NrrF. Temporal regulation of these sRNAs suggests a regulatory role in controlling functions necessary for survival, and may be important for phenotypes often associated with altered growth rates, such as biofilm formation or intracellular survival. Future functional studies will be needed to understand how these regulatory sRNAs contribute to gonococcal biology and pathogenesis.

Selection Pressure in the Human Adenovirus Fiber Knob Drives Cell Specificity in Epidemic Keratoconjunctivitis.

Human adenoviruses (HAdVs) contain seven species (HAdV-A to -G), each associated with specific disease conditions. Among these, HAdV-D includes those viruses associated with epidemic keratoconjunctivitis (EKC), a severe ocular surface infection. The reasons for corneal tropism for some but not all HAdV-Ds are not known. The fiber protein is a major capsid protein; its C-terminal "knob" mediates binding with host cell receptors to facilitate subsequent viral entry. In a comprehensive phylogenetic analysis of HAdV-D capsid genes, fiber knob gene sequences of HAdV-D types associated with EKC formed a unique clade. By proteotyping analysis, EKC virus-associated fiber knobs were uniquely shared. Comparative structural modeling showed no distinct variations in fiber knobs of EKC types but did show variation among HAdV-Ds in a region overlapping with the known CD46 binding site in HAdV-B. We also found signature amino acid positions that distinguish EKC from non-EKC types, and by in vitro studies we showed that corneal epithelial cell tropism can be predicted by the presence of a lysine or alanine at residue 240. This same amino acid residue in EKC viruses shows evidence for positive selection, suggesting that evolutionary pressure enhances fitness in corneal infection, and may be a molecular determinant in EKC pathogenesis. IMPORTANCE: Viruses adapt various survival strategies to gain entry into target host cells. Human adenovirus (HAdV) types are associated with distinct disease conditions, yet evidence for connections between genotype and cellular tropism is generally lacking. Here, we provide a structural and evolutionary basis for the association between specific genotypes within HAdV species D and epidemic keratoconjunctivitis, a severe ocular surface infection. We find that HAdV-D fiber genes of major EKC pathogens, specifically the fiber knob gene region, share a distinct phylogenetic clade. Deeper analysis of the fiber gene revealed that evolutionary pressure at crucial amino acid sites has a significant impact on its structural conformation, which is likely important in host cell binding and entry. Specific amino acids in hot spot residues provide a link to ocular cell tropism and possibly to corneal pathogenesis.

Consensus computational network analysis for identifying candidate outer membrane proteins from Borrelia spirochetes.

BACKGROUND: Similar to Gram-negative organisms, Borrelia spirochetes are dual-membrane organisms with both an inner and outer membrane. Although the outer membrane contains integral membrane proteins, few of the borrelial outer membrane proteins (OMPs) have been identified and characterized to date. Therefore, we utilized a consensus computational network analysis to identify novel borrelial OMPs. RESULTS: Using a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form ß-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes. CONCLUSIONS: Using a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.

Divergence of protein-coding capacity and regulation in the Bacillus cereus sensu lato group.

BACKGROUND: The Bacillus cereus sensu lato group contains ubiquitous facultative anaerobic soil-borne Gram-positive spore-forming bacilli. Molecular phylogeny and comparative genome sequencing have suggested that these organisms should be classified as a single species. While clonal in nature, there do not appear to be species-specific clonal lineages, excepting B. anthracis, in spite of the wide array of phenotypes displayed by these organisms. RESULTS: We compared the protein-coding content of 201 B. cereus sensu lato genomes to characterize differences and understand the consequences of these differences on biological function. From this larger group we selected a subset consisting of 25 whole genomes for deeper analysis. Cluster analysis of orthologous proteins grouped these genomes into five distinct clades. Each clade could be characterized by unique genes shared among the group, with consequences for the phenotype of each clade. Surprisingly, this population structure recapitulates our recent observations on the divergence of the generalized stress response (SigB) regulons in these organisms. Divergence of the SigB regulon among these organisms is primarily due to the placement of SigB-dependent promoters that bring genes from a common gene pool into/out of the SigB regulon. CONCLUSIONS: Collectively, our observations suggest the hypothesis that the evolution of these closely related bacteria is a consequence of two distinct processes. Horizontal gene transfer, gene duplication/divergence and deletion dictate the underlying coding capacity in these genomes. Regulatory divergence overlays this protein coding reservoir and shapes the expression of both the unique and shared coding capacity of these organisms, resulting in phenotypic divergence. Data from other organisms suggests that this is likely a common pattern in prokaryotic evolution.

Homologous recombination in E3 genes of human adenovirus species D.

Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1ß, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber.

Control of RNA stability by NrrF, an iron-regulated small RNA in Neisseria gonorrhoeae.

Regulation of gene expression by small noncoding RNAs (sRNAs) plays a critical role in bacterial response to physiological stresses. NrrF, a trans-acting sRNA in Neisseria meningitidis and Neisseria gonorrhoeae, has been shown in the meningococcus to control indirectly, in response to iron (Fe) availability, the transcription of genes encoding subunits of succinate dehydrogenase, a Fe-requiring enzyme. Given that in other organisms, sRNAs target multiple mRNAs to control gene expression, we used a global approach to examine the role of NrrF in controlling gonococcal transcription. Three strains, including N. gonorrhoeae FA1090, an nrrF deletion mutant, and a complemented derivative, were examined using a custom CombiMatrix microarray to assess the role of this sRNA in controlling gene expression in response to Fe availability. In the absence of NrrF, the mRNA half-lives for 12 genes under Fe-depleted growth conditions were longer than those in FA1090. The 12 genes controlled by NrrF encoded proteins with biological functions including energy metabolism, oxidative stress, antibiotic resistance, and amino acid synthesis, as well as hypothetical proteins and a regulatory protein whose functions are not fully understood.

Overreliance on the hexon gene, leading to misclassification of human adenoviruses.

The genome of human adenovirus (HAdV) D30 was sequenced in depth. Sequence assembly and analysis revealed two distinct viral sequences with identical hexon genes, which were the same as the one previously reported for HAdV-D30. However, one of the two viruses was found to be a recombinant of HAdV-D29. Exclusive reliance on serum neutralization can lead to mischaracterization of adenoviruses and miss coinfections. Whole-genome sequencing remains the gold standard for proper classification of HAdVs.

Five genome sequences of subspecies B1 human adenoviruses associated with acute respiratory disease.

Five genomes of human subspecies B1 adenoviruses isolated from cases of acute respiratory disease have been sequenced and archived for reference. These include representatives of two prevalent genomic variants of HAdV-7, i.e., HAdV-7h and HAdV-7d2. The other three are HAdV-3/16, HAdV-16 strain E26, and HAdV-3+7 strain Takeuchi. All are recombinant genomes. Genomics and bioinformatics provide detailed views into the genetic makeup of these pathogens and insight into their molecular evolution. Retrospective characterization of particularly problematic older pathogens such as HAdV-7h (1987) and intriguing isolates such as HAdV-3+7 strain Takeuchi (1958) may provide clues to their phenotypes and serology and may suggest protocols for prevention and treatment.

Genome sequence of the fish pathogen Flavobacterium columnare ATCC 49512.

Flavobacterium columnare is a Gram-negative, rod-shaped, motile, and highly prevalent fish pathogen causing columnaris disease in freshwater fish worldwide. Here, we present the complete genome sequence of F. columnare strain ATCC 49512.

Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group.

BACKGROUND: The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species. RESULTS: During the divergence of these organisms from a common "SigB-less" ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence. CONCLUSIONS: Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.

MpeR regulates the mtr efflux locus in Neisseria gonorrhoeae and modulates antimicrobial resistance by an iron-responsive mechanism.

Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.

Complete genome sequence of human adenovirus prototype 17.

As one of the first five human adenoviruses (HAdVs) to be sequenced, type 17 was important as a reference tool for comparative genomics of recently isolated HAdV pathogens in species D. HAdV-D17 was the first species D adenovirus to be sequenced and was deposited in GenBank in 1999. These genome data were not of high quality, and a redetermination of the same stock virus provides corrected data; among the differences are a length of 35,139 bp versus 35,100 bp in the original, and 160 mismatches to the original genome were found. Annotation of the coding sequences reveals 39 as opposed to 8, a finding which is important for phylogenomic studies.

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver.

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After ~8-11 months of WD feeding, dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in maternal WD (mWD) exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriaceae and Prevotellaceae were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group.

BACKGROUND: The Bacillus cereus sensu lato group consists of six species (B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the Bc species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of B. subtilis. RESULTS: Phylogenetic analysis of the Bc species-group utilizing 157 single-copy genes of the family Bacillaceae suggests that several taxonomic revisions of the genus Bacillus should be considered. Within the Bc species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the Bc species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the Bc species-group. CONCLUSIONS: Expansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in B. subtilis. Divergence of the sigma-controlled transcriptional regulons among various members of the Bc species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the Bc species-group.

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses.

BACKGROUND: Pneumonia and myocarditis are the most commonly reported diseases due to Histophilus somni, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in H. somni using traditional methods. Analyses of the genome sequences of several Pasteurellaceae species have provided insights into their biology and evolution. In view of the economic and ecological importance of H. somni, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the Pasteurellaceae. RESULTS: The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, several H. somni genes that may encode proteins involved in virulence (e.g., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor. CONCLUSIONS: Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two H. somni strains.

Computational analysis of two species C human adenoviruses provides evidence of a novel virus.

Human adenovirus C (HAdV-C) species are a common cause of respiratory infections and can occasionally produce severe clinical manifestations. A deeper understanding of the variation and evolution in species HAdV-C is especially important since these viruses, including HAdV-C6, are used as gene delivery vectors for human gene therapy and in other biotechnological applications. Here, the full-genome analysis of the prototype HAdV-C6 and a recently identified virus provisionally termed HAdV-C57 are reported. Although the genomes of all species HAdV-C members are very similar to each other, the E3 region, hexon and fiber (ten proteins total) present a wide range of identity values at the amino acid level. Studies of these viruses in comparison to the other three HAdV-C prototypes (1, 2, and 5) comprise a comprehensive analysis of the diversity and conservation within HAdV-C species. HAdV-C6 contains a recombination event within the constant region of the hexon gene. HAdV-C57 is a recombinant virus with a fiber gene nearly identical to HAdV-C6 and a unique hexon distinguished by its loop 2 motif.

Antibacterial efficacy of non-thermal atmospheric plasma against Streptococcus mutans biofilm grown on the surfaces of restorative resin composites.

The aim of this study was to evaluate the antimicrobial efficacy of non-thermal atmospheric plasma (NTAP) against Streptococcus mutans biofilms. Resin discs were fabricated, wet-polished, UV sterilized, and immersed in water for monomer extraction (37 °C, 24 h). Biofilms of bioluminescent S. mutans strain JM10 was grown on resin discs in anaerobic conditions for (37 °C, 24 h). Discs were divided into seven groups: control (CON), 2% chlorhexidine (CHX), only argon gas 150 s (ARG) and four NTAP treatments (30 s, 90 s, 120 s, 150 s). NTAP was applied using a plasma jet device. After treatment, biofilms were analyzed through the counting of viable colonies (CFU), bioluminescence assay (BL), scanning electron microscopy (SEM), and polymerase chain reaction (PCR). All NTAP-treated biofilm yielded a significant CFU reduction when compared to ARG and CON. BL values showed that NTAP treatment for 90 s, 120 s or 150 s resulted in statistically significantly lower metabolic activity when compared to the other groups. CHX displayed the lowest means of CFU and BL. SEM showed significant morphological changes in NTAP-treated biofilm. PCR indicated damage to the DNA structure after NTAP treatment. NTAP treatment was effective in lowering the viability and metabolism of S. mutans in a time-dependent manner, suggesting its use as an intraoral surface-decontamination strategy.

Transcriptional and functional analysis of the Neisseria gonorrhoeae Fur regulon.

To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies--temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)--to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.