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Biomedical subjects

J Heesemann

Publications and source records attributed to J Heesemann.

At least 91 records · Page 5Linked to original sources

Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli.

From two different specimens of a chronic prosthetic hip infection taken at an interval of 2 months a slow-growing gram-negative bacterium was isolated in pure culture. The strain grew with the typical features of a small-colony variant (SCV). 16S rRNA sequencing identified the bacterium as Escherichia coli. Biochemical characterization demonstrated multiple phenotypic alterations of a mutant carrying a defect in the heme biosynthetic pathway (Hem-): (i) catalase and nitrate reductase reactions were both negative, (ii) a negative benzidine reaction demonstrated the lack of heme-containing cytochromes, and (iii) growth stimulation under anaerobic conditions as well as gentamicin resistance indicated defective aerobic respiration. PCR and Southern hybridization demonstrated that the mutation of the SCV of E. coli was localized in the hemB gene and was most likely due to a deletion of the hemB gene. On blood agar plates revertants were recognized growing as normal-sized colonies between the dominant small colonies of the strain. Feeding experiments indicated that the revertants but not the small colonies were permeable for hemin. A strong antibody response against the infecting SCV of E. coli was found. To our knowledge, this is the first report of a Hem- E. coli strain as the etiological agent of a chronic bacterial infection.

Escherichia coli↗

Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species.

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.

Animals↗

Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH2-terminal kinase. Correlation with its inhibitory effect on tumor necrosis factor-alpha production.

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-alpha (TNFalpha) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNFalpha release, (ii) the suppressor effect on TNFalpha production, which originates from the lack of TNFalpha mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNFalpha production by inhibiting ERK1/2, p38, and JNK kinase activities.

Animals↗

[Mycoplasma hominis. A rare causative agent of acute pyelonephritis].

HISTORY AND CLINICAL FINDINGS: A 23-year-old woman was admitted with typical signs of an acute urinary tract infection: fever, pain on tapping over both renal areas and in both flanks, urgency and dysuria. She had a history of renal colic with spontaneous passage of a renal stone. INVESTIGATIONS: There was marked leukocytosis and raised C-reactive protein, leukocyturia and haematuria, but no nitrites or protein in the urine. All blood and urine cultures were sterile and renal ultrasound was unremarkable. DIAGNOSIS, TREATMENT AND COURSE: As signs and laboratory data indicated acute pyelonephritis (PN) she was treated with gyrase inhibiting antibiotics. But while symptoms improved, fever, leukocyturia and haematuria continued; no micro-organism could be demonstrated. Mycoplasma was therefore considered as a rare cause of PN. Special urine cultures then grew M. hominis, > 10(5) organisms/ml. On the basis of sensitivity tests doxycycline was administered. All symptoms quickly improved and all inflammation parameters and urine sediments became normal. CONCLUSION: In rare instances M. hominis may be isolated as the causative organism of PN. If, in cases with appropriate symptoms, routine tests fail to demonstrate the causative agent, M. hominis should be included in the differential diagnosis.

Acute Disease↗

High frequency of Yersinia antibodies in healthy populations in Finland and Germany.

Sera from 94 healthy Finnish and 100 healthy German blood donors were studied for Yersinia enterocolitica antibodies by two different techniques, enzyme immunoassay and immunoblotting, to compare the frequency of Yersinia infections in Finnish and German populations. The prevalence of Yersinia antibodies in Finland (19% and 31% by enzyme immunoassay and immunoblotting, respectively) and in Germany (33%, 43%) is relatively high and may indicate many subclinical Yersinia infections in healthy populations. Postinfectious complications, such as reactive arthritis, may follow also such subclinical infection; sensitive serological tests are important in their diagnosis.

Adolescent↗

Characterization of Escherichia coli serotype O12:K1:H7 isolates from an immunocompetent carrier with a history of spontaneous abortion and septicemia.

Escherichia coli isolates from an immunocompetent woman with a history of repeated amnion infections and spontaneous abortion were characterized. Escherichia coli were isolated from stool, blood and cervix swab samples taken over a 21-month period after the last abortion which followed septicemia during pregnancy. All samples except the last cervix swab contained isolates of serotype O12:K1:H7, which produced adhesins, P fimbriae, type I fimbriae and the iron-chelator aerobactin. Pulsed-field gel electrophoresis revealed identical Xbal restriction patterns of the O12:K1:H7 isolates, suggesting that one particular Escherichia coli strain was responsible for the severe extraintestinal infections during pregnancy. The strain was able to persist in the intestine of the woman despite antibiotic therapy.

Abortion, Spontaneous↗

Yersinia enterocolitica: a cause of chronic polyarthritis.

To investigate the role of Yersinia persistence in chronic undifferentiated arthritis, two patients who had chronic undifferentiated polyarthritis and circulating IgA and IgG antibodies to Yersinia outer proteins were studied. Immunofluorescence using antibodies directed against Yersinia adhesin A was performed on colonic and synovial tissue. Synovial tissue T cells were cloned aspecifically and screened for their proliferative responses to Yersinia enterocolitica. Furthermore, a Yersinia-specific polymerase chain reaction (PCR) was performed on synovial tissue. Both patients were found to have Yersinia antigens in colonic and synovial tissue. Y. enterocolitica-positive T-cell clones were grown from the synovial tissue: 4 CD4+ clones of 37 clones from patient 1 and 6 CD4+ clones of 53 clones from patient 2. Yersinia-specific PCR products were not detected in the synovial tissue specimens. The results support the hypothesis that an immune-mediated response to Yersinia antigens may play an important role in the pathogenesis of chronic undifferentiated arthritis.

Adhesins, Bacterial↗

Contribution of the Mn-cofactored superoxide dismutase (SodA) to the virulence of Yersinia enterocolitica serotype O8.

Enteric pathogens harbor a set of enzymes (e.g., superoxide dismutases [SOD]) for detoxification of endogenous and exogenous reactive oxygen species which are encountered during infection. To analyze the role of the Mn-cofactored SOD (SodA) in the pathogenicity of yersiniae, we cloned the sodA gene of Yersinia enterocolitica serotype O8 by complementation of an Escherichia coli sodA sodB mutant and subsequently constructed an isogenic mutant by allelic exchange. Sequence analysis revealed an open reading frame that enabled the deduction of a sequence of 207 amino acids with 85% identity to SodA of E. coli. In a mouse infection model, the sodA null mutant was strongly attenuated in comparison to its parental strain. After intravenous infection, the survival and multiplication of the mutant in the spleen and liver were markedly reduced. In contrast, inactivation of sodA had only minor effects on survival and multiplication in the gut and Peyer's patches, as could be demonstrated in the orogastric infection model. The reduction in virulence was accompanied by a low but significant increase of susceptibility of the soda mutant to bacterial killing by polymorphonuclear leukocytes (PMN) and an alteration of the intracellular chemiluminescence response of PMN. These results suggest that the resistance of Y. enterocolitica to exogenous oxygen radicals produced by phagocytes involves the Mn-cofactored SOD. The important role of sodA for the pathogenicity of Y. enterocolitica could also be due to detoxification of endogenous, metabolically produced oxygen radicals which are encountered by extracellular enteric pathogens during the invasion of the host.

Animals↗

Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis.

Suppression of the host defense is one of the hallmarks of Yersinia enterocolitica infection. This enteric pathogen resists phagocytosis and interferes with macrophage functions from an extracellular localization (oxidative-burst generation and tumor necrosis factor alpha production). In this study, we investigated the fate of the Y. enterocolitica-infected macrophage. We found that murine J774A.1 macrophages and macrophages derived from human monocytes were killed by infection with Y. enterocolitica. Analysis of cellular morphology and DNA fragmentation revealed that macrophage cell death occurs through the induction of apoptosis. A total of 92% +/- 5% (mean +/- standard deviation) of murine J774A.1 macrophages and 74% +/- 6% of human monocyte-derived macrophages underwent apoptosis upon Yersinia infection after 4 and 20 h, respectively. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone blocked completion of the Yersinia-induced apoptotic program but not the surface exposure of phosphatidylserine as an early-stage apoptotic event. Analysis of different Yersinia mutants showed that macrophage apoptosis depends on a functional Y. enterocolitica type III protein secretion system. Apoptotic cell death of macrophages was not related to the YopE-mediated cytotoxic effect of Yersinia, since disruption of actin microfilaments by a Y. enterocolitica strain expressing a restricted repertoire of yop genes, including YopE, did not result in macrophage apoptosis. Furthermore, Yersinia-induced cytotoxic alterations in epithelial HeLa cells, which are conferred by YopE, did not lead to apoptosis. Our data demonstrate for the first time that Y. enterocolitica promotes the apoptosis of macrophages, an effect which is clearly distinct from the morphological alterations mediated by Yersinia on epithelial HeLa cells.

Actins↗

Passive immunity to infection with Yersinia spp. mediated by anti-recombinant V antigen is dependent on polymorphism of V antigen.

The V antigen is a 37-kDa secreted polypeptide encoded on the 70-kb virulence plasmid of pathogenic Yersinia spp. Besides having regulatory functions, it is known to be a virulence factor and a protective antigen. DNA sequencing of the most common serotypes of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis revealed that two evolutionary distinct types of V antigen exist in Yersinia spp. One type is represented by Y. enterocolitica serotype 08 strains WA, WA-314, and NCTC 10938 (designated LcrV-YenO8); the other type comprises Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O3, O9, and O5,27 (LcrV-Yps). A hypervariable region between amino acids 225 and 232 represents the main difference between the two types. By raising monospecific antisera against both types of V antigen (anti-rVO8 and anti-rVO3), we were able to demonstrate that, in general, passive immunization of mice against a challenge with yersiniae was possible with both anti-Y. enterocolitica V antigen sera. However, anti-V antigen serum was protective only if the immunizing V antigen was the same type as the V antigen produced by the infective strain. The failure of the American V antigen type represented by Y. enterocolitica serotype O8 to protect against Yersinia spp. carrying the other V antigen type (LcrV-Yps) could be an explanation for the presence of plague foci in American countries.

Amino Acid Sequence↗

Invasion and persistence of Salmonella in human fibroblasts positive or negative for endogenous HLA B27.

OBJECTIVE: Analysis of the interaction of enteropathogenic bacteria with HLA B27 transfected murine fibroblasts showed a specific influence of HLA B27 on microbial invasiveness. This possible novel mechanism for the action of HLA B27 should be verified by using endogenous HLA B27 positive and negative human fibroblasts as a model for the direct interaction of arthritogenic bacteria and host cells. METHODS: Fibroblasts were obtained from healthy donors positive or negative for HLA B27; cultivated as monolayers; and infected with Salmonella enterica serovar enteritidis. RESULTS: Invasion and survival of bacteria in human cells was not influenced by the presence of HLA B27. Enhancement of HLA class I molecule expression by treatment of cultures with interferon gamma decreased invasion and survival of bacteria in both HLA B27 positive and negative cells. After disappearance of live bacteria lipopolysaccharide antigens persisted within cells. CONCLUSION: Endogenous HLA B27 does not modulate the direct interaction of Salmonella with human cells. Non-professional phagocytes are able to limit bacterial survival in cells, and interferon gamma accelerates killing of bacteria, but arthritogenic antigens persist after disappearance of live bacteria.

Analysis of Variance↗

The influence of HLA B27 and interferon-gamma on the invasion and persistence of yersinia in primary human fibroblasts.

To examine the role of the human HLA class I molecule B27 in the direct interaction of yersinia and human cells we performed experimental infections of constitutive HLA B27-positive and -negative human fibroblast monolayers with Yersinia enterocolitica O.3. The numbers of yersiniae invading HLA B27-positive cells were within the range of HLA B27-negative cells. Treatment of fibroblasts with interferon-gamma (IFN-gamma) to enhance HLA B27 expression did not alter invasion. IFN-gamma reduced titers of live intracellular bacteria, but not bacterial antigens, in a dose-dependent manner. Persistent infection with yersinia could be maintained for up to 10 weeks with decreasing bacterial titers and more slowly decreasing amounts of yersinial antigens in HLA B27-positive and -negative cells. Endogenous HLA B27 does not modulate the direct interaction of yersiniae with primary human fibroblasts. This is in contrast to infection of HLA B27-transfected murine cells. The protective effect of IFN-gamma for yersinia-infected cells may be important for controlling complications in patients with yersiniosis.

Adhesins, Bacterial↗

Structural and functional organization of the Yersinia pestis bacteriocin pesticin gene cluster.

The primary structure of a 2671 bp DNA fragment between the pla gene (encoding plasminogen activator) and the origin of replication of the wild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 1074 and 426 bp with opposite transcription polarities were identified on both strands. They encode a 357 aa pesticin activity protein (Pst) and a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and serve as rho-independent transcription terminator sequences for both genes. The site for the interaction with the LexA repressor of the SOS system was found in another palindromic structure preceding the pst structural gene. A deduced 39.9 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pentapeptide typical of TonB-dependent colicins (TonB box) that is necessary for the interaction with the yersiniabactin/pesticin receptor and for active energy-dependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not significantly influence the bactericidal activity of the truncated pesticin. The pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino acids. A deduced 16 kDa Pim protein has an N-terminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pl. The immunity protein was localized in the periplasmic space and in the outer-membrane fraction after its overexpression under the polymerase T7 promoter. Several other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necessary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (> 70%) identity with other colicin genes. The DNA sequence located 284 bp upstream of the pim gene showed more than 90% similarity to antisense RNA I of the ColE1 replicon. This defined the location of the pYP358 origin of ColE1-type replication.

Amino Acid Sequence↗

Translocation of Yersinia enterocolitica through an endothelial monolayer by polymorphonuclear leukocytes.

An endothelial cell monolayer grown on a microporous membrane coated with basement membrane protein matrix was used to study translocation of yersinia-infected human polymorphonuclear leukocytes (PMNs). PMNs infected with one to eight bacteria were able to translocate living yersiniae from the upper chamber to the chemoattractant-containing lower chamber. This process may contribute to extravasation and dissemination of yersiniae in the infected host.

Animals↗

Differential contribution of Yersinia enterocolitica virulence factors to evasion of microbicidal action of neutrophils.

The differential contribution of the virulence factors invasin, protein tyrosine phosphatase (YopH), cytotoxin (YopE), and adhesin (YadA) of Yersinia enterocolitica to evasion of the antibacterial activities of polymorphonuclear leukocytes (PMNs) (oxidative burst, phagocytosis, killing) was analyzed. We constructed virulence gene knockout mutants and a novel two-plasmid system allowing production and secretion of individual virulence factors. Wild-type Y. enterocolitica WA-314 harboring the virulence plasmid pYV08 resisted phagocytosis and killing by PMNs. Moreover, strain WA-314 was able to inhibit the neutrophil oxidative burst upon stimulation with opsonized zymosan independently on preincubation with normal human serum or YadA-specific serum. These phenotypic properties of strain WA-314 were differentially affected when mutants impaired in YadA production or Yop secretion were used. A more detailed analysis revealed that YopH plays the dominant role in suppression of the antibacterial action of PMNs without damaging the cells. The YopH suppressing effect could be enhanced by coproduction of YopE and YadA. The contribution of YadA is attributed to the adhesin function promoting interaction with PMNs under both opsonizing and nonopsonizing conditions. In contrast, invasin seems to mediate only opsonin-independent interaction with PMNs. Taken together, our results demonstrate that YopH, YopE, and YadA act in concert towards neutrophil attack to enable extracellular survival of Y. enterocolitica in host tissue.

Adhesins, Bacterial↗

Experimental Yersinia infection of human synovial cells: persistence of live bacteria and generation of bacterial antigen deposits including "ghosts," nucleic acid-free bacterial rods.

Yersinia enterocolitica O:3 was maintained in primary cultures of human synovial cells for 6 weeks as cultivable organisms and thereafter for 2 more weeks as antigen aggregates containing specific lipopolysaccharides (LPS). Some seemingly intact bacteria were "ghosts," bacterial rods possessing LPS but not DNA. The prolonged persistence of yersiniae, and consequently of Yersinia antigens, in synovial cells may be the cause of the maintenance of the inflammatory host responses in the joints of patients with reactive arthritis due to Yersinia infection.

Antigens, Bacterial↗

Deletion of amino acids 29 to 81 in adhesion protein YadA of Yersinia enterocolitica serotype O:8 results in selective abrogation of adherence to neutrophils.

In order to analyze the multiple functions of the yersinia adhesin YadA in more detail, we constructed an N-terminally truncated YadA protein (deletion of amino acids [aa] 29 to 81) of Yersinia enterocolitica serotype 0:8. The region aa 29 to 81 of YadA is located between the signal sequence and the amino-terminal hydrophobic domain (aa 80 to 101), which is involved in surface polymerization and collagen binding. The deletion of aa 29 to 81 (resulting in YadADelta29-81) had no effect on the well-known features of YadA such as autoagglutination, serum resistance, HEp-2 cell adherence, binding of collagen, and binding of the complement-inhibiting factor H. In contrast to this, mutant WA(pYVO8-A-Delta29-81), producing the truncated YadADelta29-81 had lost the ability to adhere to polymorphonuclear leukocytes and to induce an oxidative burst. This functional deficiency was comparable to that of a yadA-null mutant (K. Ruckdeschel, A. Roggenkamp, S. Schubert, and J. Heesemann, Infect. Immun. 64:724-733, 1996). Moreover, mutant WA(pYVO8-ADelta29-81) turned out to be attenuated in virulence comparably to the yadA-null mutant, as demonstrated with orogastrically and intravenously infected mice. In summary, this study shows that specific functions of YadA (i) can be impaired by designed mutations and (ii) are important in distinct stages of the infection process.

Adhesins, Bacterial↗