A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence-HMW1, HMW3, P30, P90, P40, and P65-colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.Mycoplasmas are parasitic bacteria with a small genome size and no peptidoglycan layer (36). Several mycoplasmas have terminal structures which enable them to adhere to the host cell surface for colonization and nutrient acquisition. The terminal structure of Mycoplasma pneumoniae, designated the attachment organelle, has been well described (19,20). It is a membrane protrusion supported by a cytoskeleton-like structure and characterized by a dense cluster of the adhesin protein known as P1 (35).Electron microscopic images have suggested that M. pneumoniae cells divide by binary fission and that the formation and migration of the attachment organelle are coordinated with the cell division process (6). However, the actual order of cell images relative to the cell cycle must be known, and information about the timing of DNA replication is required, in order to substantiate this model. In previous works we quantified and localized the chromosomal DNA through the observation of 4Ј,6Ј-diamidino-2-phenylindole (DAPI)-stained cells of Mycoplasma capricolum by fluorescence microscopy (40, 41). This technique may also be useful for examining the cell division process of M. pneumoniae, although it does not provide the required information about the position of the attachment organelle. Recently, immunofluorescence microscopy was used to study the subcellular localization of bacterial proteins (27). This technique, combined with DAPI...

Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8 T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNβ, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNβ are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages. Inflamed gastrointestinal tissue reveals evidence for HLA-B27 misfolding, ERAD, and autophagy, without acute UPR activation. A more complete picture of conditions that impact HLA-B27 folding and misfolding, the full spectrum and time course of consequences of ER stress, and critical cell types involved is needed to understand the role of HLA-B27 misfolding in spondyloarthritis pathogenesis.

SummaryAlmost four decades of research into the role of human leukocyte antigen-B27 (HLA-B27) in susceptibility to spondyloarthritis has yet to yield a convincing answer. New results from an HLA-B27 transgenic rat model now demonstrate quite convincingly that CD8 + T cells are not required for the inflammatory phenotype. Discoveries that the HLA-B27 heavy chain has a tendency to misfold during the assembly of class I complexes in the endoplasmic reticulum (ER) and to form aberrant disulfide-linked dimers after transport to the cell surface have forced the generation of new ideas about its role in disease pathogenesis. In transgenic rats, HLA-B27 misfolding generates ER stress and leads to activation of the unfolded protein response (UPR), which dramatically enhances the production of interleukin-23 (IL-23) in response to pattern recognition receptor agonists. These findings have led to the discovery of striking T-helper 17 cell activation and expansion in this animal model, consistent with results emerging from humans with spondyloarthritis and the discovery of IL23R as an additional susceptibility gene for ankylosing spondylitis. Together, these results suggest a novel link between HLA-B27 and the T-helper 17 axis through the consequences of protein misfolding, and open new avenues of investigation as well as identifying new targets for therapeutic intervention in this group of diseases.

Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta-and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated ␤-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain.Mycoplasma pneumoniae is an extracellular pathogen of the human respiratory tract (26, 46) causing histopathological changes of lung epithelial cells, usually in older children and young adults (18). A critical step in bacterial colonization of the host cells is the specific adhesion to host cell receptors, mediated by bacterial adhesins. In M. pneumoniae, the P1 adhesin (3,17,25,26) and the adhesin-related 30-kDa protein (4, 10) have been identified. Both proteins are located mainly in a tip structure that functions as the attachment organelle of the bacterium. It could be demonstrated, by nearestneighbor analysis with a hydrophilic chemical cross-linker, that the product of open reading frame 6 (ORF6) of the P1 operon (28), a 40-kDa protein and 90-kDa protein (9, 34, 50) are located in close proximity to the P1 adhesin on the cell surface (35).Scanning and transmission electron microscope analyses of M. pneumoniae cells grown on grids and pretreated with Triton X-100 revealed a rodlike tip structure and a network of filamentous strands (22,41). Krause and coworkers identified a set of high-molecular-weight proteins (HMW1 to HMW5) which play an important role in cytadherence (30,31,(51)(52)(53) and appear to be involved in formation of a cytoskeletonlike structure (38). These proteins, ...

Adherence of Mycoplasma pneumoniae to host cells requires several mycoplasmal membrane proteins and cytoskeleton-like proteins in addition to the adhesin P1, a transmembrane protein of 170 kDa. To analyse interactions of the P1 adhesin with other membrane proteins or with cytoskeleton-like proteins, cross-linking studies were performed in vivo using the permeant reagent paraformaldehyde. The cross-linked protein complex was isolated by immunoaffinity chromatography, and proteins complexed to the P1 protein were identified by immunoblot analysis followed by high mass accuracy tryptic peptide mapping using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). In addition to the P1 protein and a truncated form of the same protein, the adhesin-related 30 kDa protein, two membrane proteins of 40 and 90 kDa, the cytoskeleton-associated 65 kDa protein and two cytoskeleton-forming proteins, HMW1 and HMW3, were found to be components of the isolated protein complex. Furthermore, the cross-linked complex contained the chaperone DnaK and the E1α subunit of pyruvate dehydrogenase. In summary, it was shown that cytadherence-associated membrane proteins are located in close proximity to cytoskeleton-like proteins, suggesting a functional interaction between membrane and cytoskeleton-like proteins. DnaK might be involved in translocation of proteins from the cytoplasm to the membrane and pyruvate dehydrogenase might be a structural protein of the attachment organelle.

A spontaneous, hemadsorption-negative mutant of Mycoplasma pneumoniae lacks the cytoskeleton-forming HMW1 protein and exhibits a truncated adhesin-related 30-kDa protein. Genetic analyses revealed deletion of one nucleotide in the hmw1 gene and loss of eight repeated sequences comprising 144 nucleotides in the gene for the adhesin-related 30-kDa protein.Adherence of Mycoplasma pneumoniae to epithelial cells (cytadherence) is a highly complex multifactorial process and the prerequisite for colonization of the human respiratory tract and subsequent disease described as atypical pneumonia (2,16,32). Characterization of spontaneously cytadherence-negative mutants (9,21,22) or comparable mutants obtained after nitrosoguanidine treatment (12,13,43) resulted in the identification of proteins which are necessary for effective adherence to host cells. In addition to the adhesin P1 (170 kDa) and the adhesin-related 30-kDa protein (3,4,7,10,15,16,18,40), which are not sufficient for M. pneumoniae cytadherence, three membrane proteins of 85, 72, and 37 kDa (designated B, A, and C, respectively, by Hansen et al. [12]) and five proteinaceous components (HMW1 to -5) of the cytoskeleton-like triton shell are involved in the attachment process (17,24,30,(35)(36)(37)(38)43). These proteins are generally designated accessory proteins.We have considerable evidence that the two proteins B and C are identical with the cleavage fragments of the open reading frame 6 (ORF6) gene product (40-and 90-kDa proteins) of the P1 operon, which consists of three ORFs in the order ORF4, ORF5 (P1), ORF6 (11,17,19,21,22,26,27,35,43). Biochemical and immunological studies revealed the location of the ORF6 gene product at the tip-like organelle of M. pneumoniae in close proximity to the P1 protein (11,26,27).In M. pneumoniae the loss of cytadherence is a very frequent event. About 0.7% of colonies derived from a cytadherencepositive clone have proved hemadsorption negative (22,25). However, the molecular basis or the reasons for the high rate of mutants lacking proteins involved in cytadherence are still unclear. Until now, in only one case was the genetic basis for the loss of the proteins P1, A, B, and C in a single mutant determined (39).In this article we describe the molecular basis of mutations in the hmw1 gene and the adhesin-related 30-kDa-protein gene accompanied by loss of cytadherence.Culture conditions and isolation of a hemadsorption-negative mutant of M. pneumoniae. M. pneumoniae M129-B18 (ATCC 29342) and the spontaneous mutant M6 were grown for 48 h at 37ЊC in 137-cm 2 Roux flasks containing 120 ml of modified Hayflick medium (14). The adherent Mycoplasma colonies were scraped off into phosphate-buffered saline (PBS) (0.14 M NaCl, 0.01 M sodium phosphate [pH 7.4]). After centrifugation at 8,000 ϫ g for 10 min the cells were washed three times in PBS and the final pellets were frozen at Ϫ70ЊC.For isolation of spontaneous mutants defective in adhering to erythrocytes, for the most part the procedures of Hansen et al. (12) and Krause et al...

IntroductionPrevious observations suggest that active systemic juvenile idiopathic arthritis (sJIA) is associated with a prominent erythropoiesis gene-expression signature. The aim of this study was to determine the association of this signature with peripheral blood mononuclear cell (PBMC) subpopulations and its specificity for sJIA as compared with related conditions.MethodsThe 199 patients with JIA (23 sJIA and 176 non-sJIA) and 38 controls were studied. PBMCs were isolated and analyzed for multiple surface antigens with flow cytometry and for gene-expression profiles. The proportions of different PBMC subpopulations were compared among sJIA, non-sJIA patients, and controls and subsequently correlated with the strength of the erythropoiesis signature. Additional gene-expression data from patients with familial hemophagocytic lymphohistiocytosis (FHLH) and from a published sJIA cohort were analyzed to determine whether the erythropoiesis signature was present.ResultsPatients with sJIA had significantly increased proportions of immature cell populations, including CD34+ cells, correlating highly with the strength of the erythropoiesis signature. The erythropoiesis signature strongly overlapped with the gene-expression pattern in purified immature erythroid precursors. The expansion of immature cells was most prominently seen in patients with sJIA and anemia, even in the absence of reticulocytosis. Patients with non-sJIA and anemia did not exhibit the erythropoiesis signature. The erythropoiesis signature was found to be prominent in patients with FHLH and in a published cohort of patients with active sJIA, but not in patients with inactive sJIA.ConclusionsAn erythropoiesis signature in active sJIA is associated with the expansion of CD34+ cells, also is seen in some patients with FHLH and infection, and may be an indicator of ineffective erythropoiesis and hemophagocytosis due to hypercytokinemia.

In a pilot study, we identified Chlamydia pneumoniae in the cerebrospinal fluid by polymerase chain reaction in 5 of 10 patients with definite multiple sclerosis (MS). In a second series, 2 of 20 patients with definite MS and 3 of 17 patients with possible/probable MS or MS variants, but none of 56 patients with other neurological, diseases were polymerase chain reaction-positive. We confirm that C. pneumoniae can be found in the cerebrospinal fluid of MS patients, but our rate of positive results is lower than in a recent report.