Elaine Allan

Identification of N-Acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni.

It was demonstrated recently that there is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To char‐ acterize such glycoproteins, we identified a lectin, Soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulphate‐ polyacrylamide gel electrophoresis (SDS–PAGE), Coomassie‐stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28 kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34 kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein extracts from mutant and wild‐type strains were analysed by one‐ and two‐dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA‐reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA‐reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid‐glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of α‐linked N‐acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non‐flagellin campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain α‐linked N‐acetylgalactosamine residues.

Stress wars: the direct role of host and bacterial molecular chaperones in bacterial infection.

Since 1962, when Ferruccio Ritossa discovered new puffing patterns in the polytene chromosomes of Drosophila incubated at an elevated temperature (153), we have been aware that stress at the cellular level is answered by the production of specific gene products. These products are variously termed heat shock proteins (Hsps) or cell stress proteins and were originally identified as molecules that are produced in response to the presence of unfolded proteins within the cell (2). However, it was not until the pioneering work of the groups of Laskey, Ellis, and Georgopoulos that the relationship between the generation of correctly assembled macromolecules and the proteins that function to ensure correct assembly was established. Laskey and coworkers studied the nuclear protein nucleoplasmin, which ensures correct assembly of histones and DNA into nucleosomes. Laskey termed nucleoplasmin a molecular chaperone as it mimicked the function of a human chaperone who ensures correct interactions between people (98). Ellis and Georgopoulos studied the protein which eventually was known as chaperonin 60 (Cpn60) and which was responsible for initiating the vast flood of papers on molecular chaperones over the past two decades (58). Currently, a molecular chaperone is defined by Ellis as “one of a large and diverse group of proteins that share the property of assisting the non-covalent assembly/disassembly of other macromolecular structures, but which are not permanent components of these structures when they are performing their normal biological functions” (58). A further refinement of terminology is necessary as certain molecular chaperones are produced constitutively and the concentrations within the cell do not increase in response to stress. These proteins are defined as molecular chaperones but are not Hsps or stress proteins. Molecular chaperones, whose concentrations increase in response to stress, are both chaperones and stress proteins/heat shock proteins. The first protein-folding molecular chaperone to be discovered was Cpn60 (58). Since the identification of this protein as a molecular chaperone in 1988, many more proteins with actual or putative molecular chaperone functions have been discovered, and the term currently applies to 25 families of proteins (Table ​(Table1).1). In all three kingdoms of life molecular chaperones are classified as essential proteins, and there is significant conservation of sequences between proteins used by prokaryotes and proteins used by eukaryotes (such as thioredoxin [Trx] family members, cyclophilins, chaperonins, Hsp70, and Hsp90). Eukaryotic cells have multiple compartments (cytosol, endoplasmic reticulum, mitochondria, nucleus), and in these compartments stress-induced protein folding is known as the unfolded protein response (158). The unfolded protein responses are an important element in the integrated biology of the cell, are linked to key intracellular signaling pathways, and are now being associated with human disease states (158). Implicit in the definition of molecular chaperones was that they were intracellular proteins involved in the folding of client proteins within cellular compartments, which, because of the high protein concentration (on the order of 200 to 400 mg/ml), favor inappropriate protein-protein interactions, resulting in significant protein denaturation (58). However, it is becoming clear that many molecular chaperones can exist outside the cell and participate in nonfolding actions. TABLE 1. Eukaryotic and prokaryotic molecular chaperone and stress protein families Analysis of immune responses to bacteria in the 1970s identified what was termed a “common antigen” in many bacterial species (81). Patients infected with Mycobacterium tuberculosis or Mycobacterium leprae exhibit significant antibody responses to a 65-kDa antigen (189). Subsequent work identified this antigen (and the common antigen) as the molecular chaperone Cpn60 (193). It has now been established that a number of molecular chaperones from bacteria and protozoan parasites (Cpn60, Hsp70, and Hsp90) are (i) potent immunogens, (ii) active immunomodulators, and (iii) inducers of cross-reactive immunity and autoimmunity (176). The mammalian immune system recognizes the molecular chaperones of infecting parasites as particularly strong immunological signals, which is surprising in view of the significant homology between host and parasite proteins. This profound immune responsiveness should be useful in developing vaccines against pathogens. The Cpn60 (Hsp65) protein of M. tuberculosis has also proven to be an extremely powerful immunomodulator that is able to protect against a number of experimental autoimmune diseases in rodents, including diabetes. In a recent phase II study, immunization of individuals who had newly developed type I diabetes with a peptide derived from human Cpn60 proved to be effective in limiting the progression of this disease (146). Most researchers studying molecular chaperones work within the paradigm that these proteins are present solely in intracellular compartments. However, in 1989, just 1 year after the identification of Cpn60 as a molecular chaperone, Japanese scientists found that the protein-folding catalyst and stress protein thioredoxin was secreted by T cells from patients with a certain form of leukemia and was able to induce T cells to express one of the subunits of the interleukin-2 (IL-2) receptor (167). Subsequently, the human protein was found to be a potent chemoattractant for neutrophils, monocytes, and T lymphocytes with a unique mechanism of action (124). Since this initial discovery, a growing number of mammalian molecular chaperones have been found to be secreted onto the cell surface or into the extracellular milieu, either tissue culture fluid (with cultured cells) or biological fluids such as blood, synovial fluid, or bronchoalveolar secretions (Table ​(Table2).2). Most of these secreted proteins have agonist activity with a mammalian cell population(s), normally myeloid and lymphoid cells and/or vascular endothelial cells (VECs) (62). Although less attention has been paid to the cell-cell signaling activity of bacterial molecular chaperones, bacterial Cpn10, Cpn60, and Hsp70 have been reported to stimulate or inhibit the proinflammatory actions of myeloid cells and VECs (62). Thus, it is becoming clear that molecular chaperones are examples of moonlighting proteins, that is, proteins which have more than one function. The enzymes of glycolysis are the prototypic moonlighting proteins. For example, secreted phosphoglucoisomerase has been identified as three distinct cytokines: neuroleukin, autocrine motility factor, and differentiation and maturation mediator. This protein also acts as an implantation factor (76). Perhaps the most bizarre example of the moonlighting functions of molecular chaperones is the neurotoxin used by a hunting insect, the antlion or doodlebug, to paralyze its prey. This toxin is produced by a symbiotic bacterium, Enterobacter aerogenes, which lives in the insects saliva. It has been determined that this toxin is a molecular chaperone, Cpn60. The E. aerogenes Cpn60 is almost identical to the Escherichia coli Cpn60 protein GroEL. Surprisingly, single-residue substitutions in GroEL can change it from an inactive protein into a potent insect neurotoxin (187). TABLE 2. Molecular chaperones found on the cell surface and/or secreted by cells and/or found in extracellular fluids In the last decade there have been a number of reports which support the hypothesis that inducible molecular chaperones, produced both by bacteria and by hosts, function as intracellular, cell surface, or extracellular signals which are involved in the control of the infectious process. This suggests that infection, among other things, is a contest of stress mechanisms with a multitude of unexpected evolutionary twists and turns. In this review we focus on bacterial infection but discuss examples from eukaryotic parasites that exemplify particular mechanisms. The molecular chaperones involved in the formation of the type III secretion system, while indirectly contributing to bacterial virulence, are not included here as they have been extensively reviewed elsewhere (48).

Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers

Clostridium difficile is a major nosocomial pathogen and the main causative agent of antibiotic-associated diarrhoea. The organism produces two potent toxins, A and B, which are its major virulence factors. These are chromosomally encoded on a region termed the pathogenicity locus (PaLoc), which also contains regulatory genes, and is absent in non-toxigenic strains. Here we show that the PaLoc can be transferred from the toxin-producing strain, 630Δerm, to three non-toxigenic strains of different ribotypes. One of the transconjugants is shown by cytotoxicity assay to produce toxin B at a similar level to the donor strain, demonstrating that a toxigenic C. difficile strain is capable of converting a non-toxigenic strain to a toxin producer by horizontal gene transfer. This has implications for the treatment of C. difficile infections, as non-toxigenic strains are being tested as treatments in clinical trials.

Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile.

Background Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of ‘hypervirulent’ strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, R20291, two further CTns were described. Results CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements. Conclusions The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host.

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis.

Characterization of the low-pH responses of Helicobacter pylori using genomic DNA arrays

Helicobacter pylori is unique among bacterial pathogens in its ability to persist in the acidic environment of the human stomach. To identify H. pylori genes responsive to low pH, the authors assembled a high-density array of PCR-amplified random genomic DNA. Hybridization of radiolabelled cDNA probes, prepared using total RNA from bacteria exposed to buffer at either pH 4.0 or pH 7.0, allowed both qualitative and quantitative information on differential gene expression to be obtained. A previously described low-pH-induced gene, cagA, was identified together with several novel genes that may have relevance to the survival and persistence of H. pylori in the gastric environment. These include genes encoding enzymes involved in LPS and phospholipid synthesis and secF, encoding a component of the protein export machinery. A hypothetical protein unique to H. pylori (HP0681) was also found to be acid induced. Genes down-regulated at pH 4.0 include those encoding a sugar nucleotide biosynthesis protein, a flagellar protein and an outer-membrane protein. Differential gene expression was confirmed by total RNA slot-blot hybridization.

Clostridium difficile Modulates Host Innate Immunity via Toxin-Independent and Dependent Mechanism(s)

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI.

Mutational Analysis of Genes Encoding the Early Flagellar Components of Helicobacter pylori: Evidence for Transcriptional Regulation of Flagellin A Biosynthesis

We investigated the roles of fliF, fliS, flhB, fliQ, fliG, and fliI of Helicobacter pylori, predicted by homology to encode structural components of the flagellar basal body and export apparatus. Mutation of these genes resulted in nonmotile, nonflagellate strains. Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein. RNA slot blot hybridization showed reduced levels of flaA mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway. This is the first demonstration of a checkpoint in H. pylori flagellar assembly.

In silico analysis of sequenced strains of Clostridium difficile reveals a related set of conjugative transposons carrying a variety of accessory genes.

The human gut pathogen Clostridium difficile contains many conjugative transposons that have an array of accessory genes. In the current study, recently sequenced genomes were analyzed to identify new putative conjugative transposons. Eleven new elements in 5 C. difficile strains were identified and all had a similar structure to the previously described elements CTn1, CTn5 and CTn7 in C. difficile strain 630. Each element identified did however contain a new set of accessory genes compared with those previously reported; including those predicted to encode ABC transporters, a toxin/antitoxin system and multiple antibiotic resistance genes.

Mobile genetic elements in Clostridium difficile and their role in genome function

Approximately 11% the Clostridium difficile genome is made up of mobile genetic elements which have a profound effect on the biology of the organism. This includes transfer of antibiotic resistance and other factors that allow the organism to survive challenging environments, modulation of toxin gene expression, transfer of the toxin genes themselves and the conversion of non-toxigenic strains to toxin producers. Mobile genetic elements have also been adapted by investigators to probe the biology of the organism and the various ways in which these have been used are reviewed.