A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni

Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) C. jejuni strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (\u3e5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in C. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni

Genomic Insights into Campylobacter jejuni Virulence and Population Genetics

Campylobacter jejuni has long been recognized as a main food-borne pathogen in many parts of the world. Natural reservoirs include a wide variety of domestic and wild birds and mammals, whose intestines offer a suitable biological niche for the survival and dissemination of the organism. Understanding the genetic basis of the biology and pathogenicity of C. jejuni is vital to prevent and control Campylobacter-associated infections. The recent progress in sequencing techniques has allowed for a rapid increase in our knowledge of the molecular biology and the genetic structures of Campylobacter. Single-molecule realtime (SMRT) sequencing, which goes beyond four-base sequencing, revealed the role of DNA methylation in modulating the biology and virulence of C. jejuni at the level of epigenetics. In this review, we will provide an up-to-date review on recent advances in understanding C. jejuni genomics, including structural features of genomes, genetic traits of virulence, population genetics, and epigenetics

Multi-omics Approaches to Deciphering a Hypervirulent Strain of Campylobacter jejuni

Campylobacter jejuni clone SA recently emerged as the predominant cause of sheep abortion in the United States and is also associated with foodborne gastroenteritis in humans. A distinct phenotype of this clone is its ability to induce bacteremia and abortion. To facilitate understanding the pathogenesis of this hypervirulent clone, we analyzed a clinical isolate (IA3902) of clone SA using multi-omics approaches. The genome of IA3902 contains a circular chromosome of 1,635,045 bp and a circular plasmid of 37,174 bp. Comparative genomic analysis revealed that IA3902 is most closely related to C. jejuni NCTC11168, which is a reference strain and was previously shown to be non-abortifacient in pregnant animals. Despite the high genomic synteny and sequence homology, there are 12 variable regions (VRs) and 8,696 single-nucleotide polymorphisms and indels between the two genomes. Notably, the variable genes in the capsular polysaccharides biosynthesis and O-linked glycosylation loci of IA3902 are highly homogenous to their counterparts in C. jejuni subsp. doylei and C. jejuni G1, which are known to be frequently associated with bacteremia. Transcriptomic and proteomic profiles were conducted to compare IA3902 with NCTC11168, which revealed that the pathways of energy generation, motility, and serine utilization were significantly up-regulated in IA3902, whereas the pathways of iron uptake and proline, glutamate, aspartate, and lactate utilization were significantly down-regulated. These results suggest that C. jejuni clone SA has evolved distinct genomic content and gene expression patterns that modulate surface polysacharide structures, motilitiy, and metabolic pathways. These changes may have contributed to its hyper-virulence in abortion induction

Heterogeneous ice nucleation correlates with bulk-like interfacial water

Establishing a direct correlation between interfacial water and heterogeneous ice nucleation (HIN) is essential for understanding the mechanism of ice nucleation. Here, we study the HIN efficiency of polyvinyl alcohol (PVA) surfaces with different densities of hydroxyl groups. We find that the HIN efficiency increases with the decrease of the hydroxyl group density. By explicitly considering that interfacial water molecules of PVA films consist of ‘tightly bound water’, ‘bound water’ and ‘bulk-like water’, we reveal that ‘bulk-like water’ can be correlated directly to the HIN efficiency of surfaces. As the density of hydroxyl groups decreases, ‘bulk-like water’ molecules can rearrange themselves with a reduced energy barrier into ice due to the diminishing constraint by the hydroxyl groups on the PVA surface. Our study not only provides a new strategy on experimentally controlling HIN efficiency but also gives another perspective in understanding the mechanism of ice nucleation, i.e., the phase change efficiency of ‘bulk-like’ interfacial water of a film is a predictor for the HIN efficiency of that film

The Rho-independent transcription terminator for the porA gene enhances expression of the major outer membrane protein and Campylobacter jejuni virulence in abortion induction

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. Its porA gene encodes the major outer membrane protein (MOMP) that is abundantly expressed and has important physiological functions including a key role in systemic infection and abortion induction in pregnant animals. Despite the importance of porA in C. jejuni pathogenesis, mechanisms modulating its expression levels remain elusive. At the 3′ end of the porA transcript, there is a Rho-independent transcription terminator (named TporA in this study). Whether TporA affects the expression and function of MOMP remains unknown and is investigated in this study. Green fluorescent protein (GFP) fusion constructs with the porA promoter at the 5′ end and an intact TporA or no TporA at the 3′ end of the gfp coding sequence revealed that both transcript level of gfp and its fluorescence signals were more than 2-fold higher in the construct with TporA than the one without TporA. qRT-PCR analysis of the porA mRNA and immunoblotting detection of the MOMP protein in C. jejuni showed that disruption of TporA significantly reduced the porA transcript level and expression of the MOMP protein. mRNA decay assay demonstrated that disruption of TporA resulted in shortened transcript half-life of the upstream gfp or porA genes, indicating TporA enhances mRNA stability. In the guinea pig model, the C. jejuni construct with an interrupted TporA was significantly attenuated in abortion induction. Together these results indicate that TporA enhances the expression level of MOMP by stabilizing its mRNA and influences the virulence of C. jejuni

Susceptibility of schizophrenia and affective disorder not associated with loci on chromosome 6q in Han Chinese population

<p>Abstract</p> <p>Background</p> <p>Several linkage studies across multiple population groups provide convergent support for susceptibility loci for schizophrenia – and, more recently, for affective disorder – on chromosome 6q. We explore whether schizophrenia and affective disorder have common susceptibility gene on 6q in Han Chinese population.</p> <p>Methods</p> <p>In the present study, we genotyped 45 family trios from Han Chinese population with mixed family history of schizophrenia and affective disorder. Twelve short tandem repeat (STRs) markers were selected, which covered 102.19 cM on chromosome 6q with average spacing 9.29 cM and heterozygosity 0.78. The transmission disequilibrium test (TDT) was performed to search for susceptibility loci to schizophrenia and affective disorder.</p> <p>Results</p> <p>The results showed STRs D6S257, D6S460, D6S1021, D6S292 and D6S1581 were associated with susceptibility to psychotic disorders. When families were grouped into schizophrenia and affective disorder group, D6S257, D6S460 and D6S1021, which map closely to the centromere of chromosome 6q, were associated with susceptibility to schizophrenia. Meanwhile, D6S1581, which maps closely to the telomere, was associated with susceptibility to affective disorder. But after correction of multiple test, all above association were changed into no significance (P > 0.05).</p> <p>Conclusion</p> <p>These results suggest that susceptibility of schizophrenia and affective disorder not associated with loci on chromosome 6q in Han Chinese population.</p

Genetic Diversity and Antimicrobial Susceptibility of Campylobacter jejuni Isolates Associated with Sheep Abortion in the United States and Great Britain

Campylobacter infection is a leading cause of ovine abortion worldwide. Historically, genetically diverse Campylobacter fetus and Campylobacter jejunistrains have been implicated in such infections, but since 2003 a highly pathogenic, tetracycline-resistant C. jejuni clone (named SA) has become the predominant cause of sheep abortions in the United States. Whether clone SA was present in earlier U.S. abortion isolates (before 2000) and is associated with sheep abortions outside the United States are unknown. Here, we analyzed 54 C. jejuniisolates collected from U.S. sheep abortions at different time periods and compared them with 42 C. jejuni isolates associated with sheep abortion during 2002 to 2008 in Great Britain, using multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and array-based comparative genomic hybridization (CGH). Although clone SA (ST-8) was present in the early U.S. isolates, it was not as tetracycline resistant (19% versus 100%) or predominant (66% versus 91%) as it was in the late U.S isolates. In contrast, C. jejuni isolates from Great Britain were genetically diverse, comprising 19 STs and lacking ST-8. PFGE and CGH analyses of representative strains further confirmed the population structure of the abortion isolates. Notably, the Great Britain isolates were essentially susceptible to most tested antibiotics, including tetracycline, while the late U.S. isolates were universally resistant to this antibiotic, which could be explained by the common use of tetracyclines for control of sheep abortions in the United States but not in Great Britain. These results suggest that the dominance of clone SA in sheep abortions is unique to the United States, and the use of tetracyclines may have facilitated selection of this highly pathogenic clone

Novel genome polymorphisms in BCG vaccine strains and impact on efficacy

Bacille Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis currently used as a vaccine against tuberculosis. Global distribution and propagation of BCG has contributed to the in vitro evolution of the vaccine strain and is thought to partially account for the different outcomes of BCG vaccine trials. Previous efforts by several molecular techniques effectively identified large sequence polymorphisms among BCG daughter strains, but lacked the resolution to identify smaller changes. In this study, we have used a NimbleGen tiling array for whole genome comparison of 13 BCG strains. Using this approach, in tandem with DNA resequencing, we have identified six novel large sequence polymorphisms including four deletions and two duplications in specific BCG strains. Moreover, we have uncovered various polymorphisms in the phoP-phoR locus. Importantly, these polymorphisms affect genes encoding established virulence factors including cell wall complex lipids, ESX secretion systems, and the PhoP-PhoR two-component system. Our study demonstrates that major virulence factors are different among BCG strains, which provide molecular mechanisms for important vaccine phenotypes including adverse effect profile, tuberculin reactivity and protective efficacy. These findings have important implications for the development of a new generation of vaccines

Intestinal colonization and acute immune response in commercial turkeys following inoculation with Campylobacter jejuni constructs encoding antibiotic-resistance markers

Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, of which Campylobacter jejuni subsp. jejuni (C. jejuni) is responsible for approximately 90% of the cases. At slaughter, the ceca of commercial chickens and turkeys are the main anatomical site where C. jejuni asymptomatically colonizes. We have previously colonized commercial turkey poults with different isolates of C. jejuni and evaluated different media to best enumerate Campylobacter from intestinal samples, but the host-response is unknown in turkeys. Enumeration of Campylobacter(colony forming units (cfu)/gram of intestinal contents) can be challenging, and can be confounded if animals are colonized with multiple species of Campylobacter. In order to precisely enumerate the C. jejuni isolate used to experimentally colonize turkeys, constructs of C. jejuni (NCTC 11,168) were tagged with different antibiotic resistance markers at the CmeF locus (chloramphenicol (CjCm) or kanamycin (CjK)). We sought to examine the kinetics of intestinal colonization using the antibiotic resistant constructs, and characterize the immune response in cecal tissue of turkeys. In vitroanalysis of the tagged antibiotic-resistant constructs demonstrated no changes in motility, morphology, or adherence and invasion of INT-407 cells compared to the parent isolate NCTC 11,168. Two animal experiments were completed to evaluate intestinal colonization by the constructs. In experiment 1, three-week old poults were colonized after oral gavage for 14 days, and CjCm and CjK cfu were recovered from cecal, but not ileal contents. In experiment 2, nine-week old poults were orally inoculated with CjCm, and the abundance of CjCm cfu/g of cecal contents significantly decreased beyond 14 days after inoculation. Significant lesions were detected in CjCm colonized poults at day 2 post-colonization. Using immunohistochemistry, Campylobacter antigen was detected in between cecal villi by day 7 of CjCm colonized poults. Quantitative RT-PCR of CjCm-colonized cecal tissue demonstrated significant down-regulation of IL-1β, IL-10 and IL-13 mRNA, and significant up-regulation of IL-6, IL-8, IL-17 A, IL-22 and IFNγ mRNA on day 2, and for some on day 7 post-colonization. All differentially expressed genes were similar to mock-infected poults by day 14. These data suggest that C. jejuni induced a brief inflammatory response in the cecum of poults that quickly resolved. Results from this study provide valuable insight into host-response and persistent colonization of the turkey cecum. These findings will help to develop and test strategies to promote food safety in commercial turkeys