Dysregulation of macrophage PEPD in obesity determines adipose tissue fibro-inflammation and insulin resistance.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes.

Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice. Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91phox(-/)- mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox(-/)- mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS(-/)- mice only beyond the first week of infection. Influx of inflammatory CD11b(+) cells, granuloma formation, and serum interferon gamma levels were unimpaired in iNOS(-/)- mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.

Role of bacterial intimin in colonic hyperplasia and inflammation.

Enteropathogenic Escherichia coli (EPEC) cells adhere to gut epithelial cells through intimin alpha: the ligand for a bacterially derived epithelial transmembrane protein called the translocated intimin receptor. Citrobacter rodentium colonizes the mouse colon in a similar fashion and uses a different intimin: intimin beta. Intimin alpha was found to costimulate submitogenic signals through the T cell receptor. Dead intimin beta+ C. rodentium, intimin alpha-transfected C. rodentium or E. coli strain K12, and EPEC induced mucosal hyperplasia identical to that caused by C. rodentium live infection, as well as a massive T helper cell-type 1 immune response in the colonic mucosa. Mutation of cysteine-937 of intimin to alanine reduced costimulatory activity in vitro and prevented immunopathology in vivo. The mucosal changes elicited by C. rodentium were interferon-gamma-dependent. Immunopathology induced by intimin enables the bacteria to promote conditions that are favorable for increased microbial colonization.

Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

Point-prevalence survey of carbapenemase-producing Enterobacteriaceae and vancomycin-resistant enterococci in adult inpatients in a university teaching hospital in the UK.

Infections with carbapenemase-producing Enterobacteriaceae (CPE) and vancomycin-resistant enterococci (VRE) are associated with increased morbidity and mortality, but the carriage rates of CRE and VRE among hospital inpatients are unknown. A point-prevalence survey was conducted to determine CPE and VRE carriage rates in hospitalized adults. Eight hundred and eighteen of 960 (85.2%) adult inpatients were invited to participate in the study. Of these, 595 patients (72.7%) consented and provided specimens. Of 540 samples tested, none were positive for CPE. One hundred and thirty of 540 (24.1%) samples were VRE positive, and 34 of 40 (85%) of wards had cases. Universal screening for CPE may not be cost-effective in low-prevalence settings, but targeted screening of high-risk patients should continue. The optimal screening strategy for VRE remains to be determined, as universal screening and isolation is not feasible in the study setting.

Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes beta or gamma via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int(280)-beta) or EHEC O157:H7 (Int(280)-gamma) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1') from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1') or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int(280)-gamma and Efa-1' or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.

Antimicrobial resistance in human populations: challenges and opportunities.

Antimicrobial resistance (AMR) is a global public health threat. Emergence of AMR occurs naturally, but can also be selected for by antimicrobial exposure in clinical and veterinary medicine. Despite growing worldwide attention to AMR, there are substantial limitations in our understanding of the burden, distribution and determinants of AMR at the population level. We highlight the importance of population-based approaches to assess the association between antimicrobial use and AMR in humans and animals. Such approaches are needed to improve our understanding of the development and spread of AMR in order to inform strategies for the prevention, detection and management of AMR, and to support the sustainable use of antimicrobials in healthcare.

Erratum: Antimicrobial resistance in human populations: challenges and opportunities - ERRATUM.

[This corrects the article DOI: 10.1017/gheg.2017.4.].

The Escherichia coli gene pool.

Wild Escherichia coli are superbly adapted to survive in the intestines of their mammalian hosts and in the environment. E. coli K12 derivative (MG1655) encodes 4288 potential genes that provide the background genetic framework of this species. Particular E. coli clonal types encode additional chromosomal and extrachromosomal genes that facilitate the ability of E. coli to adapt to new environments. These additional genes are often clustered, have related functions (for example, virulence-associated genes in pathogenicity islands) and may be integrated at specific sites on the E. coli chromosome.

Comparison of the Escherichia coli K-12 genome with sampled genomes of a Klebsiella pneumoniae and three salmonella enterica serovars, Typhimurium, Typhi and Paratyphi.

The Escherichia coli K-12 genome (ECO) was compared with the sampled genomes of the sibling species Salmonella enterica serovars Typhimurium, Typhi and Paratyphi A (collectively referred to as SAL) and the genome of the close outgroup Klebsiella pneumoniae (KPN). There are at least 160 locations where sequences of >400 bp are absent from ECO but present in the genomes of all three SAL and 394 locations where sequences are present in ECO but close homologs are absent in all SAL genomes. The 394 sequences in ECO that do not occur in SAL contain 1350 (30.6%) of the 4405 ECO genes. Of these, 1165 are missing from both SAL and KPN. Most of the 1165 genes are concentrated within 28 regions of 10-40 kb, which consist almost exclusively of such genes. Among these regions were six that included previously identified cryptic phage. A hypothetical ancestral state of genomic regions that differ between ECO and SAL can be inferred in some cases by reference to the genome structure in KPN and the more distant relative Yersinia pestis. However, many changes between ECO and SAL are concentrated in regions where all four genera have a different structure. The rate of gene insertion and deletion is sufficiently high in these regions that the ancestral state of the ECO/SAL lineage cannot be inferred from the present data. The sequencing of other closely related genomes, such as S.bongori or Citrobacter, may help in this regard.

The molecular mechanisms of severe typhoid fever.

Salmonella typhi continues to cause severe disease in many parts of the world, its most feared complication being perforation of ulcerated Peyer's patches within the small intestine, leading to peritonitis with associated mortality. The pathogenesis of this process is not well understood. In this article, we present a theoretical mechanism as to how bacterial factors and host immunological mediators within infected tissue might contribute to the observed intestinal pathology, and propose that necrosis of the Peyer's patches observed in typhoid is caused by a mechanism similar to the Shwartzman and Koch reactions.

Intimin and the host cell--is it bound to end in Tir(s)?

Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, intimin and Tir. Some key remaining questions include whether intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate intimin-Tir association. Recent data support the hypothesis that the formation of a stable intimin-Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Immune response following oral administration of cholera toxin B subunit to HIV-1-infected UK and Kenyan subjects.

OBJECTIVE: To determine the effect of HIV-1 infection on immunoglobulin (Ig) G and IgA antibody response and circulating antibody forming cell response to oral immunization with the B subunit of cholera toxin. DESIGN: Healthy UK volunteers, and HIV-1-positive UK and Kenyan volunteers at different clinical stages of HIV-1 infection received two oral immunizations. CD4+ T cells, serum beta 2-microglobulin and neopterin were measured as surrogate markers of disease stage, and correlated with immunization response. METHODS: Serum antitoxin IgG and IgA measured by enzyme-linked immunosorbent assay and antitoxin IgG, IgA and IgM antibody-forming cells detected by enzyme-linked immunospot assay at different times after two oral immunizations. RESULTS: UK HIV-positive volunteers (mean CD4+ T cell count, 52 x 10(6)/l) responded poorly to primary and booster immunization. HIV-infected Kenyans (752 x 10(6)/l CD4+ T cells) had a significant primary and booster antibody response, whereas those with a mean CD4+ T cell count 186 x 10(6)/l had an insignificant primary, but significant booster response. Two oral immunizations induced antibody responses in HIV-positive Kenyan groups (who may have prior immunity from exposure to environmental bacterial toxins) of similar or greater magnitude to healthy UK volunteers. CONCLUSIONS: Mucosal immunization may recall immune memory and be of benefit in early and moderately advanced clinical HIV disease. The findings have important clinical implications in that mucosally targeted vaccines are potentially useful in this group of patients.

Gene expression and the development of live enteric vaccines.

Rationally attenuated live Salmonella vaccines provide good protection against homologous challenge and can act as carriers of heterologous antigens. However, the optimum expression system for each heterologous antigen will need to be established individually. This will ensure that the antigen in question is produced at appropriate levels, in the correctly folded conformation, within or at the surface of the carrier cell, and can therefore elicit the optimal immune response.

Phase and antigenic variation--the impact on strategies for bacterial vaccine design.

Many pathogens have the ability to vary the antigenic composition of surface-associated antigens. Often, this variation is mediated by the regulation of gene expression. By varying its antigenicity, the pathogen is able to avoid host immune responses more efficiently; however, this makes the design of vaccines against pathogens that exhibit antigenic variation difficult. In this review, we use the pathogenic Neisseria as an example of antigenically variable bacteria and discuss some attempts to overcome the problems of vaccine design posed by such organisms.

Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101.

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).

Cloning and characterisation of the serC and aroA genes of Yersinia enterocolitica, and construction of an aroA mutant.

A gene library of Yersinia enterocolitica 8081 was constructed in the cosmid vector pHC79. Recombinants containing the aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, were identified by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. All six recombinant plasmids which complemented aroA also complemented the serC mutation in E. coli K-12 strain KL282. Tn5 mutagenesis suggested serC encoding 3-phosphoserine aminotransferase was the proximal gene in an operon with aroA. The nucleotide sequence of a 3-kb HindII-EcoRV fragment encoding the two genes was determined. The serC and aroA open reading frames contain 362 and 428 codons, respectively, and the deduced amino acid sequences share 78% and 81% homology, respectively, with the corresponding E. coli genes. Sequence inspection revealed no obvious terminators or promoters in the intergenic region. The cloned Y. enterocolitica aroA gene was inactivated in vitro and reintroduced into the parental Y. enterocolitica 8081 strain using the suicide vector pJM703.1. Stable aroA insertion mutants of Y. enterocolitica were isolated.