A novel ex vivo set-up for dynamic long-term characterization of processes on mucosal interfaces by confocal imaging and simultaneous cytokine measurements.

We present a novel organ-explant imaging system for easy and cost-effective extended-time observation of host-pathogen interactions at mucosal interfaces. Data are complemented by parallel cytokine measurements at high temporal resolution. The set-up is based on a custom-built reusable organ chamber compatible with standard microscopes. Luminal and basal side of the explanted mucosa are connected to separate channels for optimized incubation and cytokine measurements, oxygen is provided via membrane oxygenation. Dynamic imaging with confocal microscopy permits a detailed analysis of the dynamics of pathogen-host cell interactions at the mucosal interface and the neighbouring tissue at high resolution. The system can be applied to various hollow organs with few modifications. Here we present first applications to study representative infections such as uropathogenic Escherichia coli (UPEC) infections in the urinary bladder or amoebiasis of the colon by using mouse organs. We show (i) intracellular bacteria in UPEC infections, (ii) phagocytic events on tissue during infection, as well as (iii) tissue invasion of virulent protozoans into epithelia. The versatility of this system and its higher degree of control in comparison with both traditional explant microscopy and in vivo two photon imaging solutions make it a valuable and easy-to-use addition to other current imaging techniques.

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production.

In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.

A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism.

Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.

Modulation of Rho GTPases and the actin cytoskeleton by YopT of Yersinia.

Pathogenic Yersinia species evade the innate cellular immune response by injecting antihost effector proteins (Yersinia outer proteins, Yops) into host cells through a type III secretion (TTS) apparatus. One of the six effector Yops, YopT, inactivates the small GTPase RhoA by removing the geranylgeranylated C-terminal cysteine. This cleavage results in release of RhoA from the cell membrane and subsequently in blockage of stress fiber formation. Thus YopT impairs cellular functions associated with cytoskeleton rearrangements.

Prevalence of extended-spectrum beta-lactamases in isolates of the Enterobacter cloacae complex from German hospitals.

In 2002, 119 isolates of the Enterobacter cloacae complex were collected randomly from 11 German laboratories nationwide. Antibiotic susceptibilities were tested by disk-diffusion tests according to CLSI guidelines, and MICs were determined using Etests. PCRs were performed to amplify all TEM and SHV, and most CTX-M and OXA beta-lactamase genes. PCR products were sequenced to identify the precise extended spectrum beta-lactamase (ESBL) types. Isoelectric focusing (IEF) and PM/PML Etests were used to confirm production of the respective ESBLs. According to susceptibility tests and CLSI criteria, 49 (40%) isolates were resistant to extended-spectrum cephalosporins. Seven (5.8%) isolates were positive in at least one of the PCR assays. Sequencing identified production of TEM-1 beta-lactamase genes by three (2.9%) isolates, and ESBL genes of the CTX-M and SHV beta-lactamase families by five (4.2%) isolates. IEF confirmed the production of beta-lactamases in the expected pI ranges of the respective ESBLs, and four of the five ESBL-producers were detected using the PM/PML Etest. All ESBL-producing isolates showed co-resistance to sulphonamides.

[Bacterial infections of the lungs in mucoviscidosis patients]. / Mukoviszidose-Patienten werden immer erwachsener. Der ewige Kampf gegen die Pseudomonaden.

The pathogenesis of lung infections in patients with mucoviscidosis (cystic fibrosis, CF) is multifactorial. Both host- and pathogen-related factors are involved. The most important germ in terms of progression and pulmonary damage is Pseudomonas aeruginosa. However, the clinical relevance of other CF-typical pathogens has not yet been determined with certainty, and must be assessed on the basis of the clinical presentation of the individual case. To ensure optimal patient management, a CF-specific microbiological diagnostic work-up is mandatory. Since ever more patients now survive into adulthood, the problems associated with chronic infection are gaining in importance. In light of increasing multiresistance, the use of inhalation antibiotics, as well as combined antibiotic treatment, is becoming more and more determinative for the therapeutic outcome.

[Bacterial infections following injections and infusion caused by errors of hygiene--how to avoid them]. / Infektionen nach Injektion und Infusion: So vermeiden Sie Hygienefehler.

Even minor medical interventions, such as injections, are associated with the risk of life-threatening infections--both in the doctor's office and hospital settings. Medical personnel in particular must always assume that they may be contaminated by facultative pathogenic, but potentially highly virulent, germs, although they themselves remain asymptomatic. Against this background, hygienic hand disinfection and proper skin disinfection are important hygiene measures for the prevention of infections, in particular in the case of invasive interventions. Strict attention must be paid to asepsis when preparing for injections and infusions. Sterile items must be protected against contamination. With regard to compliance with and application of hygiene standards, every physician must be an exemplary role model. Furthermore, all medical professional groups must receive appropriate training in hygiene management.

The Yersinia Ser/Thr protein kinase YpkA/YopO directly interacts with the small GTPases RhoA and Rac-1.

Pathogenic bacteria of the genus Yersinia counteract host defense by interfering with eukaryotic signal transduction pathways. YpkA of Yersinia pseudotuberculosis shares significant homology with eukaryotic Ser/Thr protein kinases, is translocated into the host cell and has been shown to be an essential virulence factor in a mouse infection model. In this study, we identify the small GTPases RhoA and Rac-1 as eukaryotic binding partners of YpkA and its homolog YopO of Yersinia enterocolitica. We demonstrate that the interaction is independent of phosphorylation of YpkA and nucleotide loading state of the GTPases. The interaction with RhoA and Rac-1 might provide an important clue to how YpkA interferes with eukaryotic signaling on a molecular level.

HLA-B27 as a relative risk factor in ankylosing enthesopathy in transgenic mice.

HLA-B27 is a risk factor for several human diseases through a mechanism that is not yet understood. This article describes a naturally occurring joint disease in laboratory mice, ANKENT. ANKENT begins with mild inflammation and culminates in irreversible stiffening of the ankle and/or tarsal joints in one or both hind paws. The macroscopic and histologic features of ANKENT, its relationship to age, gender, and environment, and some immunologic aspects are considered. With respect to genetics, it is demonstrated that an HLA-B27 transgene is a relative risk factor for ANKENT. Its impact depends on the H-2 haplotype, reaching a relative risk value of 9.4 for C57Bl/10, H-2b males (p < 0.025). Several features of ANKENT are reminiscent of human AS: joint pathology, age and gender distribution, the presence of non-MHC as well as MHC risk factors (including HLA-B27), and the suspicion that environmental factors are involved.

Immunohistological characterization of the cellular immune response against Yersinia enterocolitica in mice: evidence for the involvement of T lymphocytes.

Previous work from this laboratory has demonstrated that cloned T lymphocytes from spleens of Yersinia-infected mice can transfer immunity against Y. enterocolitica into naive animals. In this study, we investigated the cellular immune response to parenteral infection of Yersinia-resistant C57 BL/6 mice with the highly virulent Y. enterocolitica strain WA of serotype O:8 employing immunohistological methods. In the course of the infection the spleen and the liver were the organs most extensively affected. Histologically, three different patterns of inflammatory reactions could be observed: (i) small non-pyogenic granuloma-like lesions (in the liver only), (ii) microabscesses lacking a sharp outline, and (iii) larger abscesses disclosing a distinct cellular border (spleen and liver). Immunohistologically, Y. enterocolitica was detectable within abscesses but not in the small granuloma-like lesions present in the liver. CD11b/18 positive cells (= Mac-1-antigen expressed on macrophages, monocytes, granulocytes and NK-cells) could be shown in Yersinia-induced lesions. The number of these cells correlated with the extent of tissue alterations induced by Y. enterocolitica. More strikingly, we were able to demonstrate for the first time that both CD4 (helper) and CD8 (cytotoxic) T lymphocytes are present in Yersinia-induced lesions. In summary, we could demonstrate for the first time that granuloma-like lesions can be induced by Y. enterocolitica. Moreover, we supported our recent study suggesting that T lymphocytes are probably involved in the immune response against Y. enterocolitica in mice.

Antibody activity against lipopolysaccharides, lipid A and proteins from Enterobacteriaceae in patients with chronic inflammatory liver diseases.

These studies are concerned with detection of circulating antibodies against various defined enterobacterial antigens in patients with chronic inflammatory liver diseases such as chronic hepatitis type B (n = 46), chronic active hepatitis (CAH) of autoimmune type (n = 10), alcoholic cirrhosis (n = 24) and primary biliary cirrhosis (PBC) (n = 24) as well as in healthy individuals (n = 39). Anti-LPS and anti-lipid A were determined by hemolytic and hemagglutination assay. Immunoblot technique was used to investigate the antibody activity against plasmid encoded proteins from Yersinia enterocolitica. Persistent titers of anti-LPS up to serum dilution 1:32.768 were found with hemolytic and hemagglutination assay in patients with alcoholic cirrhosis or PBC and in healthy control. In contrast nearly 50% of patients with chronic hepatitis B had no hemolytic antibodies against the two LPS E. coli serotypes at the time of liver biopsy. Anti-lipid A was detectable in 58% of patients with alcoholic cirrhosis but in low titers in less than 10% in the other groups (p less than 0.001). Alcoholic cirrhosis was also associated with a high frequency of IgG and IgA antibodies against plasmid encoded proteins from Yersinia enterocolitica. The data indicate that the O-polysaccharides as strong antigens are physiologically exposed to the immune system while lipid A and enterobacterial proteins are solely immunogenic under abnormal conditions.

Experimental Yersinia enterocolitica infection in rodents: a model for human yersiniosis.

Yersinia enterocolitica infection in humans causes a broad spectrum of diseases ranging from acute bowel disease to extraintestinal manifestations such as reactive arthritis, erythema nodosum and uveitis. During the last decade a fascinating part of the molecular biology of the pathogenicity of human pathogenic Yersinia species has been unraveled. Pathogenicity factors such as protein tyrosine phosphatase, protein kinase, thrombin- and collagen-binding factors have been identified and characterized on the molecular level. In contrast to many animal models for human enteropathogenic microorganisms, experimental Y. enterocolitica infection in rodents resembles yersiniosis in humans and thus offers extraordinary opportunities to study the sequential steps of the infectious process. Rabbits are suitable animals in which to study Yersinia-induced enteritis (enterotoxin-mediated) and the humoral immune response after oral infection. The role of Peyer's patches (PP) in the entry of enteropathogenic Yersinia species has been elucidated in mice and rabbits. M cells are probably the primary target cells of invading Yersiniae. Surprisingly, after penetration of the mucosal epithelial cell layer Yersinia bacilli were visualized to be exclusively extracellular in PP tissue. Obviously neutrophils within PP were unable to phagocytize the invading microorganisms. Presently, it is not clear how the microorganisms disseminate from PP into lymph nodes, spleen, liver and lung of mice where they form abscesses and granuloma-like lesions. Immunohistologically the involvement of macrophages and T cells could be demonstrated in Yersinia-induced lesions of mice. Direct evidence for the role of T cells and cytokine-activated macrophages in the host defense reaction against a primary Yersinia infection in mice could be obtained from experiments including adoptive transfer of Yersinia-specific T cells and in vivo neutralization of TNF-alpha and IFN-gamma. The experimental rat model turned out to be a suitable model for studying Yersinia-induced aseptic arthritis. Lewis- and SHR rats proved to be arthritis-susceptible. Arthritogenicity of Yersinia for rats appeared to be restricted to Y. enterocolitica of serotype 08 and correlated with the virulence potential of this serotype. Surprisingly, expression of YadA, the collagen-binding factor, was not necessary for arthritis induction. A close association between both susceptibility to arthritis induction and Yersinia infection could be demonstrated in various rat strains. Depletion of alpha/beta T-cell receptor (alpha beta-TCR)-positive T cells by treatment with alpha beta-TCR-specific antibody revealed that T cells were required for clearance of the pathogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Virulence-associated fyuA/irp2 gene cluster of Yersinia enterocolitica biotype 1B carries a novel insertion sequence IS1328.

The fyuA/irp2 gene cluster, which is part of the Yersinia pestis pigmentation (pgm) locus encoding genes involved in iron uptake and virulence, is present in all pesticin-sensitive bacteria. In Y. enterocolitica biotype 1B strains (serotypes O8, O20, O21), the fyuA/irp2 gene cluster carries an insertion of a novel repetitive sequence, IS1328. It was also found in the genome of Y. enterocolitica O5 (biotype 1A) and O13 (biotype 1B), but not in pesticin-sensitive Y. pseudotuberculosis O1 and Escherichia coli Phi. The 1353-bp repetitive element has 12-bp perfect inverted terminal repeats. A single open reading frame is capable of encoding a 334-amino acid polypeptide. IS1328 DNA has high homology with the DNA sequences located downstream of the aggR gene from the enteroaggregative E. coli (EAggEC), to the region of the R751 plasmid flanking Tn501, to the sequence following the merR gene of S. marcescens pDU1358 plasmid, and to the sequences of K. pneumoniae plasmid pCFF04. The putative polypeptide has 36.4% identity with the transposase encoded by the Coxiella burnetii IS1111a insertion sequence. The IS1328 insertion sequence could be responsible for the deletions of the fyuA/irp2 gene cluster observed in Y. enterocolitica O8 and could represent a member of a new group of widely distributed repetitive elements.

Representational difference analysis uncovers a novel IS10-like insertion element unique to pathogenic strains of Yersinia enterocolitica.

The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species.

Characterization of a novel unique restriction-modification system from Yersinia enterocolitica O:8 1B.

Genetic manipulations with enteropathogenic Yersinia enterocolitica O:8 are complicated by the presence of an efficient PstI-like YenI restriction-modification (R-M) system. We have characterized the YenI R-M system in Y. enterocolitica O:8, biotype 1B. A 5039 bp DNA fragment of the pSAK2 recombinant plasmid carrying the yenI locus was used to determine the nucleotide sequence. DNA sequence analysis identified a single 2481 bp open reading frame (ORF) that encodes an 826 amino acid large polypeptide having an apparent molecular mass of 93 kDa. The N-terminal part of the YenI ORF has 45 and 40% identity to PstI and BsuI methyltransferases (MTases), respectively; while the C-terminal part depicts 55 and 45% identity to endonucleases (ENases) of both isoschyzomeric enzymes. The yenI gene was cloned into pT7-5 plasmid and has been shown to encode a single polypeptide of expected molecular mass. A specific recognition sequence, typical to the type II R-M systems and single peptide organization, typical to type IV R-M systems, make YenI unique among known restriction-modification systems. We have constructed a truncated recombinant variant of YenI enzyme, which conserved only MTase activity, and that can be applied to YenI methylation of the DNA to be transformed into Y. enterocolitica O:8 biotype 1B strains.

Dissection of the Yersinia enterocolitica virulence plasmid pYVO8 into an operating unit and virulence gene modules.

The structural genes encoding the Yop proteins of Yersinia enterocolitica are scattered around on the virulence plasmid (pYV). The genes which are required for transactivation, secretion and translocation of the Yopos are encoded in one cluster known as the lcr-region of pYV. After the introduction of an additional SalI restriction site into pYV of Y. enterocolitica serotype O8, we were able to clone and isolate the whole lcr-region on the mobilizable low copy vector pSUP102. Analysis of this construct in a plasmidless WA-strain showed that all Yops being encoded inside the lcr-region (YopN, YopB, YopD and the V-antigen) were secreted into the culture supernatant. Moreover, this lcr-fragment was able to promote secretion of other Yops encoded by a second recombinant plasmid. Thus the translocation and function of single Yops can be studied.

Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8.

The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100.

Insertion sequence IS100 was localized on a 9.5-kb plasmid of Yersinia pestis and was shown to be specific for Y. pestis and serotype I strains of Y. pseudotuberculosis. The nucleotide sequence of IS100 isolated from this plasmid was determined. The element, which was flanked by 5-bp direct repeats, contained 1953 bp including imperfect inverted terminal repeats of 52 and 61 bp long (43 bp were identical). Two open reading frames encoding potential polypeptides of 340 and 252 amino acids were identified on one DNA strand. Nucleotide sequence as well as deduced polypeptide sequences of IS100 were homologous to those of IS21, IS232 and IS640.

Characterization of the integration site of Yersinia high-pathogenicity island in Escherichia coli.

The high-pathogenicity island (HPI) of virulent Yersiniae consists of (i) a functional core encoding for biosynthesis and uptake of the siderophore yersiniabactin and (ii) a 5- to 13-kb AT-rich region of unknown function. This Yersinia HPI has been shown to be widely distributed among different pathotypes of Escherichia coli. In this study, the insertion site of the HPI was defined in three different E. coli strains: The enteroaggregative E. coli (EAggEC) strain 17-2, the uropathogenic (UPEC) E. coli strain 536, and the probiotic E. coli DSM6601. We demonstrated that in all three E. coli isolates the HPI is associated with the asnT tRNA (5'-extremity) and truncated in the AT-rich region (3'-extremity) since the 17-bp direct repeat (DR) of the asn tRNA that flanks the HPI in Yersinia is missing in E. coli. Moreover, in comparison to the HPI-negative E. coli K-12 strain, a uniform deletion must have taken place in the E. coli chromosome adjacent to the 3'-border of the HPI.