Peadar O'Gaora

Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.

Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

The role of a stress‐response protein in Salmonella typhimurium virulence

We recently described the use of selective transposon mutagenesis to generate a series of a virulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat‐shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated Mr of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature‐sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.

Progesterone-Regulated Changes in Endometrial Gene Expression Contribute to Advanced Conceptus Development in Cattle

The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 (P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time (FABP, DGAT2, and MSTN) or duration (CRYGS) of expression pattern for genes that contribute to the composition of histotroph.

Changes in the Endometrial Transcriptome During the Bovine Estrous Cycle: Effect of Low Circulating Progesterone and Consequences for Conceptus Elongation

In cattle, elevated concentrations of circulating progesterone (P4) in the immediate postconception period are associated with advanced conceptus development, while low P4 is implicated as a causative factor in low pregnancy rates observed in dairy cows. This study aimed to: 1) describe the transcriptional changes that occur in the bovine endometrium during the estrous cycle, 2) determine how elevated P4 affects these changes, 3) identify if low P4 alters the expression of these genes, and 4) assess the impact that low P4 has on conceptus development. Relatively few differences occurred in endometrial gene expression during the early luteal phase of the estrous cycle (Day 5 vs. 7), but comparison of endometria from more distant stages of the luteal phase (Day 7 vs. 13) revealed large transcriptional changes, which were significantly altered by exogenous supplementation of P4. Induction of low circulating P4 altered the normal temporal changes in gene expression, and these changes were coordinate with a delay in the down-regulation of the PGR from the LE and GE. Altered endometrial gene expression induced by low P4 was associated with a reduced capacity of the uterus to support conceptus development after embryo transfer on Day 7. In conclusion, the present study provides clear evidence that the temporal changes in the transcriptome of the endometrium of cyclic heifers are sensitive to circulating P4 concentrations in the first few days after estrus. Under low P4 conditions, a suboptimal uterine environment with reduced ability to support conceptus elongation is observed.

Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells

Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter‐ and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low‐ and high‐adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low‐adhesive derivatives were completely different from the one found in the high‐adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low‐adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low‐and high‐adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence

The Neisseris meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin‐requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB‐dependent receptors of Gram‐negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB‐dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin‐supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.

Conceptus-Induced Changes in the Endometrial Transcriptome: How Soon Does the Cow Know She Is Pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen from Mycobacterium tuberculosis.

Protein glycosylation has an important influence on a broad range of molecular interactions in eukaryotes, but is comparatively rare in bacteria. Several antigens from Mycobacterium tuberculosis, the causative agent of human tuberculosis, have been identified as glycoproteins on the basis of lectin binding, or by detailed structural analysis. By production of a set of alkaline phosphatase (PhoA) hybrid proteins in a mycobacterial expression system, the peptide region required for glycosylation of the 19 kDa lipoprotein antigen from M.tuberculosis was defined. Mutagenesis of two threonine clusters within this region abolished lectin binding by PhoA hybrids and by the 19 kDa protein itself. Substitution of the threonine residues also resulted in generation of a series of smaller forms of the protein as a result of proteolysis. In a working model to account for these observations, we propose that the role of glycosylation is to regulate cleavage of a proteolytically sensitive linker region close to the acylated N‐terminus of the protein.

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Identification of a Mycobacterium tuberculosis Gene That Enhances Mycobacterial Survival in Macrophages

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.