Andreas Roggenkamp

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins

The non‐fimbrial adhesins, YadA of enteropathogenic Yersinia species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct ‘lollipop’‐shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N‐terminal oval head domain is followed by a putative coiled‐coil rod and terminated by a C‐terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled‐coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right‐ (Yersinia) or left‐handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.

Toll-Like Receptor 2 Participates in Mediation of Immune Response in Experimental Pneumococcal Meningitis

Heterologous expression of Toll-like receptor (TLR)2 and CD14 in Chinese hamster ovary fibroblasts was reported to confer responsiveness to pneumococcal peptidoglycan. The present study characterized the role of TLR2 in the host immune response and clinical course of pneumococcal meningitis. Pneumococcal infection of mice caused a significant increase in brain TLR2 mRNA expression at both 4 and 24 h postchallenge. Mice with a targeted disruption of the TLR2 gene (TLR2−/−) showed a moderate increase in disease severity, as evidenced by an aggravation of meningitis-induced intracranial complications, a more pronounced reduction in body weight and temperature, and a deterioration of motor impairment. These symptoms were associated with significantly higher cerebellar and blood bacterial titers. Brain expression of the complement inhibitor complement receptor-related protein y was significantly higher in infected TLR2−/− than in wild-type mice, while the expression of the meningitis-relevant inflammatory mediators IL-1β, TNF-α, IL-6, macrophage-inflammatory protein (MIP)-2, inducible NO synthase, and C3 was similar in both genotypes. We first ectopically expressed single candidate receptors in HEK293 cells and then applied peritoneal macrophages from mice lacking TLR2 and/or functional TLR4 for further analysis. Overexpression of TLR2 and TLR4/MD-2 conferred activation of NF-κB in response to pneumococcal exposure. However, pneumococci-induced TNF-α release from peritoneal macrophages of wild-type and TLR2/functional TLR4/double-deficient mice did not differ. Thus, while TLR2 plays a significant role in vivo, yet undefined pattern recognition receptors contribute to the recognition of and initiation of the host immune defense toward Streptococcus pneumoniae infection.

Analysis of chaperone-dependent Yop secretion/translocation and effector function using a mini-virulence plasmid of Yersinia enterocolitica

We have constructed a mini-pYV plasmid (pTTSS) harboring the Yersinia type three secretion system (TTSS) and the adhesin yadA on a low-copy vector. Using this system we could demonstrate for the first time that YopO, YopP, YopM, and YopQ do not require any of the known or orphan chaperones for efficient secretion/translocation. Y. enterocolitica harboring pTTSS, (WA-C(pTTSS)) was able to secrete and translocate single Yop effector proteins in trans. WA-C(pTTSS) proved to be stable and secretion of Yops was Ca2+ and temperature dependent as is the case for the parental strain. This shows that all genes necessary for translocation and expression of the Ca(2+)-dependent phenotype are contained within the cloned region. In contrast to previously published multiple yop mutants which were constructed by sequential deletion of yops, our system which harbors only the TTSS region without yops, chaperones, and unknown ORFs can be sequentially complemented with yops and sycs of choice. WA-C(pTTSS) was able to translocate YopE, YopM and YopT into HeLa cells as demonstrated by Western blotting. Translocation of YopE and YopT was strictly dependent on the presence of their respective chaperones, whereas YopM did not require a chaperone for translocation. WA-C(pTTSS) harboring yopT and sycT was shown to translocate active YopT by demonstrating modification of the small GTP-binding protein RhoA. This shows for the first time that RhoA modification is strictly dependent on YopT and does not require additional effector Yops. WA-C(pTTSS) harboring YopP was shown to induce apoptosis. This system is ideal to study chaperone-dependent Yop secretion/ translocation without the background of other effector Yops (YopE, YopM, YopO, YopP, YopT, YopH), chaperones (SycE, SycH, SycT) and unknown ORFs. In addition this system can secrete heterologous proteins fused to the N-terminal secretion/translocation domain of YopE.

Pulmonary toxoplasmosis in bone marrow transplant recipients: report of two cases and review.

Toxoplasma gondii may cause disseminated disease in bone marrow transplant (BMT) recipients. Pulmonary toxoplasmosis in BMT patients is rarely described. Mortality rates of >90% have been previously reported. Since pulmonary toxoplasmosis is extremely difficult to diagnose, it is very often detected only at autopsy. Two cases of pulmonary toxoplasmosis in BMT recipients that were diagnosed by visualization of T. gondii tachyzoites in bronchoalveolar lavage fluid and by a new semi-nested PCR method amplifying 18S rRNA from bronchoalveolar lavage fluid are presented, and the literature on pulmonary toxoplasmosis in BMT patients is reviewed.

Yersinia enterocolitica‐mediated translocation of defined fusion proteins to the cytosol of mammalian cells results in peptide‐specific MHC class I‐restricted antigen presentation

Yersinia enterocolitica delivers a set of effector proteins [Yersinia outer proteins (Yop)] into the cytosol of target cells to modulate host cell signal transduction pathways required for the extracellular survival of the bacterium. Secretion and subsequent translocation of Yop across the eukaryotic cell membrane are achieved via a type III secretion system. About 50u2009–u2009100 amino acids of the N terminus of Yop are required for chaperone‐directed secretion and translocation. In this study, it is demonstrated by immunoblot analysis of Yersinia‐infected cultured epithelial cells that one ot these proteins, YopE, can serve as a molecular carrier to deliver protein fragments of the heterologous p60 antigen of Listeria monocytogenes into the cytosol of target cells. T cell activation assays revealed that the observed type III‐mediated antigen translocation led to a p60 peptide‐specific MHC class I‐restricted antigen presentation. Efficient translocation and antigen presentation were strictly dependent on the co‐localized expression of hybrid YopE‐p60 proteins and the YopE‐specific chaperone SycE. These results suggest that the Yersinia type III secretion system may serve as an attractive tool for antigen delivery in Yersinia‐based live vaccines to induce cellular immune responses.

Cloning and characterization of the gene encoding periplasmic 2', 3'-cyclic phosphodiesterase of yersinia enterocolitica O:8

The gene encoding periplasmic 2,3-cyclic phosphodiesterase in Yersinia enterocolitica O:8 (designated cpdB), was cloned and expressed in Escherichia coli. This enzyme enables Y. enterocolitica to grow on 2,3-cAMP as a sole source of carbon and energy. Sequencing and analysis of a 3 kb ECO:RI fragment containing the cpdB gene revealed an open reading frame of 1179 bp, corresponding to a protein with a molecular mass of 71 kDa. The first 25 amino acid residues show features of a typical prokaryotic signal sequence. The predicted molecular mass of the mature peptide is therefore in agreement with the molecular mass estimated by SDS gel electrophoresis (68 kDa). The putative cpdB promoter region contains two possible -10 and -35 regions. Furthermore, the 5 untranslated region contains sequences with significant homology to the cyclic AMP-cyclic AMP receptor protein binding site and the sigma(28) consensus. This region is interrupted by an enterobacterial repetitive intergenic consensus (ERIC) sequence. Deletion of the ERIC element from the cpdB promoter region had no effect on cpdB expression. In the 3 untranslated region, a possible rho-independent transcriptional terminator was identified. The deduced amino acid sequence of the Y. enterocolitica CpdB protein shows 76% identity with CpdB of Salmonella typhimurium and E. coli. CpdB of Y. enterocolitica is exported to the periplasmic space. An isogenic Y. enterocolitica cpdB mutant strain, constructed by allelic exchange, was no longer able to grow on 2,3-cAMP as sole source of carbon and energy. The CpdB mutant showed no significant change in virulence in an oral and intravenous mouse infection model.

Prospective study on nontuberculous mycobacteria in patients with and without cystic fibrosis.

Recent reports indicated an increase of nontuberculous mycobacteria (NTM) in cystic fibrosis (CF) patients. However, it is still a matter of discussion whether criteria for diagnosis of NTM pulmonary infection established by the American Thoracic Society (ATS) are applicable for CF patients. We determined incidence and prevalence of NTM in CF patients and non-CF patients without HIV infection, and validity of ATS criteria for CF patients. Over a period of 2xa0years, 1,251 respiratory samples were investigated for mycobacteria from 91 CF and 162 non-CF patients. For all patients with NTM recovery, we reviewed clinical charts and determined outcome for up to 2xa0years. Incidence and prevalence for repeated recovery of NTM were higher in CF patients, but not significantly. CF patients with repeated recovery of NTM met clinical and bacteriological ATS criteria, but radiographic criteria were not met. Treated CF patients showed favorable clinical outcomes, as opposed to untreated patients. We propose a modified definition for diagnosis and hence treatment of NTM pulmonary infection in CF patients.

Distribution of the outer membrane haem receptor protein ChuA in environmental and human isolates of Escherichia coli

ChuA, the haem receptor of Escherichia coli, is thought to contribute to the pathogenicity of E. coli strains causing extraintestinal infections. We investigated the prevalence and distribution of chuA in E. coli analysing 304 strains from different origins. 30% of E. coli strains isolated from the environment and about 70% of E. coli strains isolated from human sources carried chuA. No difference in chuA prevalence was found between commensals isolated from the intestine of healthy volunteers and isolates from extraintestinal infections. Our results indicate that ChuA might be involved in the colonization of human hosts.

Evaluation of the ICT Malaria Pf test for rapid post-mortem diagnosis of Plasmodium falciparum malaria in corpses examined for forensic reasons

Abstract To test the diagnostic value of a rapid and simple immunochromatographic test (ICT) based on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) for post-mortem examination, blood samples from 30 consecutive corpses were analysed by ICT and Giemsa-stained blood films. Compared to microscopy, ICT had 100% sensitivity and 100% specificity even after a considerable time had passed between the presumed time of death and testing or after prolonged storage of whole blood samples. The ICT yielded positive results for four travellers who had returned from Kenya and died from Pl. falciparum malaria. The ICT might therefore serve as an additional tool for rapid malaria diagnosis, especially in non-endemic countries where experience with microscopic malaria detection is limited.

Mechanisms of Yersinia enterocolitica evasion of the host innate immune response by V antigen.

Our data demonstrate a new ligand specificity of TLR2, since LcrV is the first known secreted and non-lipidatedvirulence-associated protein of a Gram-negative bacterium utilizing TLR2 for cell activation. We conclude that yersiniae might exploit host innatepattern recognition molecules and defense mechanisms to evade the host immune response.