Evolution of mitosome metabolism and invasion-related proteins in Cryptosporidium.

BACKGROUND: The switch from photosynthetic or predatory to parasitic life strategies by apicomplexans is accompanied with a reductive evolution of genomes and losses of metabolic capabilities. Cryptosporidium is an extreme example of reductive evolution among apicomplexans, with losses of both the mitosome genome and many metabolic pathways. Previous observations on reductive evolution were largely based on comparative studies of various groups of apicomplexans. In this study, we sequenced two divergent Cryptosporidium species and conducted a comparative genomic analysis to infer the reductive evolution of metabolic pathways and differential evolution of invasion-related proteins within the Cryptosporidium lineage. RESULTS: In energy metabolism, Cryptosporidium species differ from each other mostly in mitosome metabolic pathways. Compared with C. parvum and C. hominis, C. andersoni possesses more aerobic metabolism and a conventional electron transport chain, whereas C. ubiquitum has further reductions in ubiquinone and polyisprenoid biosynthesis and has lost both the conventional and alternative electron transport systems. For invasion-associated proteins, similar to C. hominis, a reduction in the number of genes encoding secreted MEDLE and insulinase-like proteins in the subtelomeric regions of chromosomes 5 and 6 was also observed in C. ubiquitum and C. andersoni, whereas mucin-type glycoproteins are highly divergent between the gastric C. andersoni and intestinal Cryptosporidium species. CONCLUSIONS: Results of the study suggest that rapidly evolving mitosome metabolism and secreted invasion-related proteins could be involved in tissue tropism and host specificity in Cryptosporidium spp. The finding of progressive reduction in mitosome metabolism among Cryptosporidium species improves our knowledge of organelle evolution within apicomplexans.

Complete Genome Sequences for Three Chromosomes of the Burkholderia stabilis Type Strain (ATCC BAA-67).

We report here the complete annotated genome sequence of the Burkholderia stabilis type strain ATCC BAA-67. There were three circular chromosomes with a combined size of 8,527,947 bp and G+C composition of 66.4%. These characteristics closely resemble the genomes of other sequenced members of the Burkholderia cepacia complex.

Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens.

BACKGROUND: Cyclospora cayetanensis is an apicomplexan that causes diarrhea in humans. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. In this study we sequenced the genome of C. cayetanensis and inferred its metabolism and invasion components based on comparative genomic analysis. RESULTS: The genome organization, metabolic capabilities and potential invasion mechanism of C. cayetanensis are very similar to those of Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. Unlike Eimeria spp., there are no active LTR-retrotransposons identified in C. cayetanensis. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions. CONCLUSIONS: Our findings indicate that C. cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. Amino acid metabolism and post-translation modifications of proteins are some major differences between C. cayetanensis and other apicomplexans. The whole genome sequence data of C. cayetanensis improve our understanding of the biology and evolution of this major foodborne pathogen and facilitate the development of intervention measures and advanced diagnostic tools.

Malignant Transformation of Hymenolepis nana in a Human Host.

Neoplasms occur naturally in invertebrates but are not known to develop in tapeworms. We observed nests of monomorphic, undifferentiated cells in samples from lymph-node and lung biopsies in a man infected with the human immunodeficiency virus (HIV). The morphologic features and invasive behavior of the cells were characteristic of cancer, but their small size suggested a nonhuman origin. A polymerase-chain-reaction (PCR) assay targeting eukaryotes identified Hymenolepis nana DNA. Although the cells were unrecognizable as tapeworm tissue, immunohistochemical staining and probe hybridization labeled the cells in situ. Comparative deep sequencing identified H. nana structural genomic variants that are compatible with mutations described in cancer. Invasion of human tissue by abnormal, proliferating, genetically altered tapeworm cells is a novel disease mechanism that links infection and cancer.

Genetic similarities between Cyclospora cayetanensis and cecum-infecting avian Eimeria spp. in apicoplast and mitochondrial genomes.

BACKGROUND: Cyclospora cayetanensis is an important cause for diarrhea in children in developing countries and foodborne outbreaks of cyclosporiasis in industrialized nations. To improve understanding of the basic biology of Cyclospora spp. and development of molecular diagnostic tools and therapeutics, we sequenced the complete apicoplast and mitochondrial genomes of C. cayetanensis. METHODS: The genome of one Chinese C. cayetanensis isolate was sequenced using Roche 454 and Illumina technologies. The assembled genomes of the apicoplast and mitochondrion were retrieved, annotated, and compared with reference genomes for other apicomplexans to infer genome organizations and phylogenetic relationships. Sequence variations in the mitochondrial genome were identified by comparison of two C. cayetanensis nucleotide sequences from this study and a recent publication. RESULTS: The apicoplast and mitochondrial genomes of C. cayetanensis are 34,155 and 6,229 bp in size and code for 65 and 5 genes, respectively. Comparative genomic analysis showed high similarities between C. cayetanensis and Eimeria tenella in both genomes; they have 85.6% and 90.4% nucleotide sequence similarities, respectively, and complete synteny in gene organization. Phylogenetic analysis of the genomic sequences confirmed the genetic similarities between cecum-infecting avian Eimeria spp. and C. cayetanensis. Like in other coccidia, both genomes of C. cayetanensis are transcribed bi-directionally. The apicoplast genome is circular, codes for the complete machinery for protein biosynthesis, and contains two inverted repeats that differ slightly in LSU rRNA gene sequences. In contrast, the mitochondrial genome has a linear concatemer or circular mapping topology. Eight single-nucleotide and one 7-bp multiple-nucleotide variants were detected between the mitochondrial genomes of C. cayetanensis from this and recent studies. CONCLUSIONS: The apicoplast and mitochondrial genomes of C. cayetanensis are highly similar to those of cecum-infecting avian Eimeria spp. in both genome organization and sequences. The availability of sequence data beyond rRNA and heat shock protein genes could facilitate studies of C. cayetanensis biology and development of genotyping tools for investigations of cyclosporiasis outbreaks.

vanG element insertions within a conserved chromosomal site conferring vancomycin resistance to Streptococcus agalactiae and Streptococcus anginosus.

Three vancomycin-resistant streptococcal strains carrying vanG elements (two invasive Streptococcus agalactiae isolates [GBS-NY and GBS-NM, both serotype II and multilocus sequence type 22] and one Streptococcus anginosus [Sa]) were examined. The 45,585-bp elements found within Sa and GBS-NY were nearly identical (together designated vanG-1) and shared near-identity over an ~15-kb overlap with a previously described vanG element from Enterococcus faecalis. Unexpectedly, vanG-1 shared much less homology with the 49,321-bp vanG-2 element from GBS-NM, with widely different levels (50% to 99%) of sequence identity shared among 44 related open reading frames. Immediately adjacent to both vanG-1 and vanG-2 were 44,670-bp and 44,680-bp integrative conjugative element (ICE)-like sequences, designated ICE-r, that were nearly identical in the two group B streptococcal (GBS) strains. The dual vanG and ICE-r elements from both GBS strains were inserted at the same position, between bases 1328 and 1329, within the identical RNA methyltransferase (rumA) genes. A GenBank search revealed that although most GBS strains contained insertions within this specific site, only sequence type 22 (ST22) GBS strains contained highly related ICE-r derivatives. The vanG-1 element in Sa was also inserted within this position corresponding to its rumA homolog adjacent to an ICE-r derivative. vanG-1 insertions were previously reported within the same relative position in the E. faecalis rumA homolog. An ICE-r sequence perfectly conserved with respect to its counterpart in GBS-NY was apparent within the same site of the rumA homolog of a Streptococcus dysgalactiae subsp. equisimilis strain. Additionally, homologous vanG-like elements within the conserved rumA target site were evident in Roseburia intestinalis. Importance: These three streptococcal strains represent the first known vancomycin-resistant strains of their species. The collective observations made from these strains reveal a specific hot spot for insertional elements that is conserved between streptococci and different Gram-positive species. The two GBS strains potentially represent a GBS lineage that is predisposed to insertion of vanG elements.

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

Novel paramyxovirus associated with severe acute febrile disease, South Sudan and Uganda, 2012.

In 2012, a female wildlife biologist experienced fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, and a sore throat shortly after returning to the United States from a 6-week field expedition to South Sudan and Uganda. She was hospitalized, after which a maculopapular rash developed and became confluent. When the patient was discharged from the hospital on day 14, arthralgia and myalgia had improved, oropharynx ulcerations had healed, the rash had resolved without desquamation, and blood counts and hepatic enzyme levels were returning to reference levels. After several known suspect pathogens were ruled out as the cause of her illness, deep sequencing and metagenomics analysis revealed a novel paramyxovirus related to rubula-like viruses isolated from fruit bats.

Phylogenetic and ecologic perspectives of a monkeypox outbreak, southern Sudan, 2005.

Identification of human monkeypox cases during 2005 in southern Sudan (now South Sudan) raised several questions about the natural history of monkeypox virus (MPXV) in Africa. The outbreak area, characterized by seasonally dry riverine grasslands, is not identified as environmentally suitable for MPXV transmission. We examined possible origins of this outbreak by performing phylogenetic analysis of genome sequences of MPXV isolates from the outbreak in Sudan and from differing localities. We also compared the environmental suitability of study localities for monkeypox transmission. Phylogenetically, the viruses isolated from Sudan outbreak specimens belong to a clade identified in the Congo Basin. This finding, added to the political instability of the area during the time of the outbreak, supports the hypothesis of importation by infected animals or humans entering Sudan from the Congo Basin, and person-to-person transmission of virus, rather than transmission of indigenous virus from infected animals to humans.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1(T) and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1(T) and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1(T) and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1(T) maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Characterization of novel Brucella strains originating from wild native rodent species in North Queensland, Australia.

We report on the characterization of a group of seven novel Brucella strains isolated in 1964 from three native rodent species in North Queensland, Australia, during a survey of wild animals. The strains were initially reported to be Brucella suis biovar 3 on the basis of microbiological test results. Our results indicated that the rodent strains had microbiological traits distinct from those of B. suis biovar 3 and all other Brucella spp. To reinvestigate these rodent strains, we sequenced the 16S rRNA, recA, and rpoB genes and nine housekeeping genes and also performed multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA). The rodent strains have a unique 16S rRNA gene sequence compared to the sequences of the classical Brucella spp. Sequence analysis of the recA, rpoB, and nine housekeeping genes reveals that the rodent strains are genetically identical to each other at these loci and divergent from any of the currently described Brucella sequence types. However, all seven of the rodent strains do exhibit distinctive allelic MLVA profiles, although none demonstrated an amplicon for VNTR 07, whereas the other Brucella spp. did. Phylogenetic analysis of the MLVA data reveals that the rodent strains form a distinct clade separate from the classical Brucella spp. Furthermore, whole-genome sequence comparison using the maximal unique exact matches index (MUMi) demonstrated a high degree of relatedness of one of the seven rodent Brucella strains (strain NF 2653) to another Australian rodent Brucella strain (strain 83-13). Our findings strongly suggest that this group of Brucella strains isolated from wild Australian rodents defines a new species in the Brucella genus.

The phylogenetics and ecology of the orthopoxviruses endemic to North America.

The data presented herein support the North American orthopoxviruses (NA OPXV) in a sister relationship to all other currently described Orthopoxvirus (OPXV) species. This phylogenetic analysis reaffirms the identification of the NA OPXV as close relatives of "Old World" (Eurasian and African) OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. The natural reservoir host(s) for many of the described OPXV species remains unknown although a clear virus-host association exists between the genus OPXV and several mammalian taxa. The hypothesized host associations and the deep divergence of the OPXV/NA OPXV clades depicted in this study may reflect the divergence patterns of the mammalian faunas of the Old and New World and reflect a more ancient presence of OPXV on what are now the American continents. Genes from the central region of the poxvirus genome are generally more conserved than genes from either end of the linear genome due to functional constraints imposed on viral replication abilities. The relatively slower evolution of these genes may more accurately reflect the deeper history among the poxvirus group, allowing for robust placement of the NA OPXV within Chordopoxvirinae. Sequence data for nine genes were compiled from three NA OPXV strains plus an additional 50 genomes collected from Genbank. The current, gene sequence based phylogenetic analysis reaffirms the identification of the NA OPXV as the nearest relatives of "Old World" OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. Additionally, the substantial genetic distances that separate the currently described NA OPXV species indicate that it is likely that many more undescribed OPXV/NA OPXV species may be circulating among wild animals in North America.

The detection of monkeypox in humans in the Western Hemisphere.

BACKGROUND: During May and June 2003, an outbreak of febrile illness with vesiculopustular eruptions occurred among persons in the midwestern United States who had had contact with ill pet prairie dogs obtained through a common distributor. Zoonotic transmission of a bacterial or viral pathogen was suspected. METHODS: We reviewed medical records, conducted interviews and examinations, and collected blood and tissue samples for analysis from 11 patients and one prairie dog. Histopathological and electron-microscopical examinations, microbiologic cultures, and molecular assays were performed to identify the etiologic agent. RESULTS: The initial Wisconsin cases evaluated in this outbreak occurred in five males and six females ranging in age from 3 to 43 years. All patients reported having direct contact with ill prairie dogs before experiencing a febrile illness with skin eruptions. We found immunohistochemical or ultrastructural evidence of poxvirus infection in skin-lesion tissue from four patients. Monkeypox virus was recovered in cell cultures of seven samples from patients and from the prairie dog. The virus was identified by detection of monkeypox-specific DNA sequences in tissues or isolates from six patients and the prairie dog. Epidemiologic investigation suggested that the prairie dogs had been exposed to at least one species of rodent recently imported into the United States from West Africa. CONCLUSIONS: Our investigation documents the isolation and identification of monkeypox virus from humans in the Western Hemisphere. Infection of humans was associated with direct contact with ill prairie dogs that were being kept or sold as pets.