Complete genome sequence of the universal killer Salmonella enterica Serovar Typhimurium UK-1 (ATCC 68169)

The Salmonella enterica serovar Typhimurium strain UK-1 exhibits the highest invasion and virulence attributes among the most frequently studied strains. S. Typhimurium UK-1 has been used as the foundation for developing recombinant vaccines and has been used extensively on virulence and colonization studies in chickens and mice. We describe here the complete genome sequence of S. Typhimurium UK-1. Comparative genomics of Salmonella Typhimurium will provide insight into factors that determine virulence and invasion.     

Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.     

Identification of Specific Gene Sequences Conserved in Contemporary Epidemic Strains of Salmonella enterica

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.     

Short-Term Signatures of Evolutionary Change in the Salmonella enterica Serovar Typhimurium 14028 Genome

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis in humans and a typhoid-like disease in mice and is often used as a model for the disease promoted by the human-adapted S. enterica serovar Typhi. Despite its health importance, the only S. Typhimurium strain for which the complete genomic sequence has been determined is the avirulent LT2 strain, which is extensively used in genetic and physiologic studies. Here, we report the complete genomic sequence of the S. Typhimurium strain 14028s, as well as those of its progenitor and two additional derivatives. Comparison of these S. Typhimurium genomes revealed differences in the patterns of sequence evolution and the complete inventory of genetic alterations incurred in virulent and avirulent strains, as well as the sequence changes accumulated during laboratory passage of pathogenic organisms.The genomes of related bacteria can differ in three ways: (i) gene content, where one bacterial species or strain harbors genes absent from the other organism; (ii) nucleotide substitutions within largely conserved DNA sequences, which can result in amino acid changes in orthologous proteins, form pseudogenes, and promote distinct expression patterns of genes present in the two organisms; and (iii) changes in gene arrangement, caused by inversions and translocations. These differences have been observed not only across bacterial species but also among strains belonging to the same species. Recent genomic analyses have revealed that many bacterial pathogens of humans are virtually monomorphic (1) and exhibit very limited sequence diversity, raising questions about the nature of the genetic changes governing distinct behaviors. Furthermore, several bacterial pathogens that have been subjected to extensive passage in the laboratory display altered virulence characteristics, but the genetic basis for these alterations remains largely unknown. Here, we address both of these questions by determining and analyzing the genome sequences of closely related isolates of Salmonella enterica serovar Typhimurium, a Gram-negative pathogen that has been used as a preeminent model to investigate basic genetic mechanisms (2, 8, 46, 59), as well as the interaction between bacterial pathogens and mammalian hosts (11, 41).The genus Salmonella is divided into two species: Salmonella bongori and Salmonella enterica, which together comprise over 2,300 serovars differing in host specificity and the disease conditions they promote in various hosts. For example, S. enterica serovar Typhi is human restricted and causes typhoid fever, whereas serovar Typhimurium is a broad-host-range organism that causes gastroenteritis in humans and a typhoid-like disease in mice. Although the complete genome sequences of 15 Salmonella enterica strains are available, there is only a single representative of S. Typhimurium—strain LT2 (31). Despite its wide application in genetic analysis, strain LT2 is highly attenuated for virulence in both in vitro and in vivo assays (52, 56), leading many investigators to use other S. Typhimurium isolates to examine the genetic basis for bacterial pathogenesis (11, 14, 16).Over 300 virulence genes (3, 5, 47) have already been identified in Salmonella enterica serovar Typhimurium 14028 (now termed S. enterica subsp. enterica serovar Typhimurium ATCC 14028), which is a descendant of CDC 60-6516, a strain isolated in 1960 from pools of hearts and livers of 4-week-old chickens (P. Fields, personal communication). Whereas strain 14028 has been typed as LT2, a designation based on phage sensitivity (27), the two strains were isolated from distinct sources decades apart, which makes their genealogy and exact relationship obscure. A derivative of the original 14028 strain with a rough colony morphology (due to changes in O-antigen expression) was designated 14028r to distinguish it from the original smooth strain, renamed 14028s, and was used in a genetic screen for Salmonella virulence genes because it retained lethality for mice and the ability to survive within murine macrophages. Strain 14028 was also used for the identification of Salmonella genes that were specifically expressed during infection of a mammalian host (30). Both 14028 and LT2 possess a 90-kb virulence plasmid promoting intracellular replication and systemic disease (14), but they differ in their prophage contents, as is often the case among S. Typhimurium strains (12, 13).To identify the individual changes that differentiate S. Typhimurium strains and to assess the nature of variation that arises during laboratory storage and passage, we determined the genome sequence of strain 14028s. This genome was then used as a reference for sequencing its progenitors, including the original source strain CDC 60-6516 and the earliest smooth and rough variants. Our analysis uncovered the genomic differences that arose during the past decades of laboratory cultivation and showed that derivatives with different virulence potentials can follow distinct patterns of sequence evolution.     

Identification of GtgE,a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.     

Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71

This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium strains to express and deliver VP1 of enterovirus 71 (EV71) as a vaccination strategy to prevent EV71 infection in mice. When orally administered to BALB/c mice, both attenuated carrier strains, CNP101 and SL7207, were able to efficiently invade livers and spleens, while only the virulence plasmid-carrying strain SL7207 persisted for more than 30 days in these organs. A recombinant in vivo-regulated promoter expression plasmid expressing VP1 antigen of EV71 was constructed. The expression of the VP1, directed by the pagC promoter, in attenuated Salmonella was confirmed by Western blot hybridization. Both humoral and cellular immune responses were elicited in mice by oral immunization with such Salmonella-based VP1 vaccines. We evaluated the protective efficacy of the vaccines in mice using a maternal immunization protocol. With a lethal challenge, ICR newborn mice born to dams immunized with Salmonella-based VP1 vaccine showed a 50-60% survival; in contrast, none of the mice in the control group survived the challenge. Our data indicated that Salmonella-based VP1 subunit vaccines are a promising vaccine strategy in the prevention of EV71 infection.     

Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis.     

Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.     

Characterization and Differential Gene Expression between Two Phenotypic Phase Variants in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10(-4) per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10(-6) per cell per generation. Two highly virulent S. Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the Salmonella pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.     

Virulence of 32 Salmonella strains in mice

Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies.     

猪霍乱沙门氏菌C500株△crp△asd缺失株平衡致死载体系统的构建及鉴定

猪霍乱沙门氏菌C500株是用化学方法致弱、用于预防仔猪副伤寒的弱毒疫苗株,虽具有较好的免疫原性,但仍有一定的残余毒力。为了研制更加安全并保持C500株良好免疫原性的弱毒株,及将C500开发为适于粘膜免疫的疫苗活载体,本文构建了猪霍乱沙门氏菌C500株△crp△asd双缺失株平衡致死载体系统。首先构建含缺失320bp的crp(cAMP受体蛋白)基因与蔗糖敏感基因(sacB)的重组自杀性质粒,与C500接合转移,两步法筛选无抗性的△crp缺失株,用PCR证实基因组crp基因的缺失突变。用同样方法在crp缺失株基础上构建asd(天冬氨酸β-半乳糖脱氢酶)基因缺失株。该缺失株生长必需外源DAP(二氨基庚二酸)。进一步鉴定△crp缺失株的表型、生长特性、毒力等,结果表明△crp△asd缺失株构建成功。△crp△asd缺失株可以用来作为宿主载体平衡致死系统来高效表达外源基因,为深入研究以C500株为载体的口服多价疫苗奠定了基础。     

Draft Genome Sequence of Salmonella enterica Serovar Typhimurium ST1660/06, a Multidrug-Resistant Clinical Strain Isolated from a Diarrheic Patient

Salmonella enterica serovar Typhimurium is one of the most prevalent serovars of Salmonella that causes human gastroenteritis. Here, we report the draft genome sequence of the S. Typhimurium multidrug-resistant strain ST1660/06. Comparative genomic analysis unveiled three strain-specific genomic islands that potentially confer the multidrug resistance and virulence of the strain.     

Genome-wide identification of novel genomic islands that contribute to Salmonella virulence in mouse systemic infection

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV- tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117–STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118–STM3121 were identified as novel virulence-associated genes within the pheV- tRNA-located genomic island. These results indicate that a Salmonella -specific pheV- tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.     

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature-tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.     

Determinants of bluetongue virus virulence in murine models of disease

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.     

Evaluation of Brucella abortus S19 vaccine strains by bacteriological tests, molecular analysis of ery loci and virulence in BALB/c mice.

Two Brucella abortus S19 commercial vaccine strains used for vaccination against brucellosis in India and three S19 strains available as international reference were examined by microbiological assays and molecular analysis of the ery loci involved in erythritol metabolism, and tested for residual virulence in BALB/c mice. According to the sensitivity to penicillin and i-erythritol, the five strains tested had the phenotypic characteristics of strain S19. However, on culture medium containing i-erythritol, all strains developed spontaneous i-erythritol resistant colonies at mutation rates ranging from 1.42x10(-2) to 1.33x10(-6). The S19 characteristic 702 bp deletion in the erythrulose 1-phosphate dehydrogenase gene of the ery locus was present only in the three reference strains but not in the two commercial vaccines. Both commercial strains and one of the reference strains showed reduced virulence in BALB/c mice. The presence or absence in S19 strains of the 702 bp deletion in the ery locus had no correlation with either the rates of spontaneous mutation to erythritol resistance or the residual virulence in mice.     

Distinct polymorphisms in a single herpesvirus gene are capable of enhancing virulence and mediating vaccinal resistance

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek’s disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.