Renate Rosengarten

In Vitro Cell Invasion of Mycoplasma gallisepticum

ABSTRACT The ability of the widespread avian pathogen Mycoplasma gallisepticum to invade cultured human epithelial cells (HeLa-229) and chicken embryo fibroblasts (CEF) was investigated by using the gentamicin invasion assay and a double immunofluorescence microscopic technique for accurate localization of cell-associated mycoplasmas. The presence of intracellular mycoplasmas in both cell lines was clearly demonstrated, with organisms entering the eukaryotic cells within 20 min. Internalized mycoplasmas have the ability to leave the cell, but also to survive within the intracellular space over a 48-h period. Frequencies of invasion were shown to differ between the two cell lines, but were also considerably dependent on the mycoplasma input population. Of the prototype strain R, a low-passage population in artificial medium, Rlow, was capable of active cell invasion, while a high-passage population, Rhigh, showed adherence to but nearly no uptake into HeLa-229 and CEF. By passaging Rlow and Rhigh multiple times through HeLa-229 cells, the invasion frequency was significantly increased. Taken together, these findings demonstrate that M. gallisepticumhas the capability of entering nonphagocytic host cells that may provide this pathogen with the opportunity for resisting host defenses and selective antibiotic therapy, establishing chronic infections, and passing through the respiratory mucosal barrier to cause systemic infections.

Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

ABSTRACT A family of abundant surface proteins (Vpmas [variable proteins ofMycoplasma agalactiae]) undergoing phase variation inM. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboringvpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes,vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. ThevpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. ThevpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by thevsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein

ABSTRACT A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed inEscherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.

Host-pathogen interactions in mycoplasma pathogenesis: Virulence and survival strategies of minimalist prokaryotes

Despite their very small genomes mycoplasmas are successful pathogens of man and a wide range of animal hosts. Because of the lack of effective therapeutics and vaccines, mycoplasma diseases continue to be a significant problem for public health as well as livestock production with major socio-economic consequences worldwide. Recent outbreaks and epidemiological studies predict that the incidence of human and animal mycoplasma diseases might increase which indicates the urgent need to develop new approaches for prevention and therapy. Development of such reagents, however, requires a solid understanding of the molecular biology of mycoplasma infections. Knowledge in this field has considerably increased during the past decade since new techniques have been developed and adapted to mycoplasmas that allow these organisms to be studied at the molecular level. Research on the two human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium of which the genome sequences have recently been completed as well as the substantial number of studies carried out on the AIDS-associated mycoplasmas, Mycoplasma penetrans and Mycoplasma fermentans, has led the way, but a number of animal mycoplasmas are becoming increasingly appreciated as models for the study of the molecular basis of mycoplasma diseases. This review summarizes and highlights some of the recent findings concerning the molecular interactions that occur between pathogenic mycoplasmas and their hosts, both the common strategies as well as some unique approaches evolved by particular mycoplasma pathogens, including adherence to and uptake into non-phagocytic host cells, as well as mechanisms of escaping the host immune system.

Gliding mutants of Mycoplasma mobile: relationships between motility and cell morphology, cell adhesion and microcolony formation

The present study characterizes gliding motility mutants of Mycoplasma mobile which were obtained by UV irradiation. They were identified by their abnormal colony shapes in 0.1% agar medium, showing a reduced number of satellite colonies compared to the wild-type. A total of ten mutants were isolated based on their colony phenotype. Using dark-field and electron microscopy, two classes of mutants, group I and group II, were defined. Cells of group I mutants had irregular, flexible and sometimes elongated head-like structures and showed a tendency to aggregate. Neither binding to glass nor gliding motility was observed in these mutants. Cells of group II mutants were rather spherical in shape, with the long axis reduced to 80% and the short axis enlarged to 120% of that of wild-type cells, respectively. Their gliding speed was 20% faster than that of wild-type cells. Three of the ten mutants remained unclassified. Mutant m6 had a reduced binding activity to glass and a reduced gliding motility with 50% of the speed of the wild-type strain. The ability of wild-type and mutant colonies to adsorb erythrocytes was found to correlate with the binding activity required for gliding, indicating that mycoplasma gliding depends on cytadherence-associated components. Finally, the ability to form microcolonies on surfaces was shown to correlate with the gliding activity, suggesting a certain role of gliding motility in the parasitic life-cycle of mycoplasmas.

Characterization of methicillin-resistant Staphylococcus spp. carrying the mecC gene, isolated from wildlife

OBJECTIVES A recently identified mecA homologue, mecC, in methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from humans and different animal hosts. The aim of this study was to determine antimicrobial resistance and provide molecular characterization of MRSA and methicillin-resistant non-Staphylococcus aureus staphylococci (MRnSA) isolated from wildlife that carried the gene mecC. METHODS Five S. aureus and one coagulase-negative Staphylococcus isolate displaying phenotypic oxacillin resistance, but not recognized with conventional PCR for mecA, were further characterized by a polyphasic approach. The presence of mecC in all isolates was determined using specific PCR. PCR targeting Panton-Valentine leucocidin (PVL) genes of MRSA was performed. MRSA isolates were genotyped by spa typing and multilocus sequence typing. All isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing. 16S rDNA sequence analysis for MRnSA identification was performed. Antimicrobial susceptibility testing was performed for all isolates. RESULTS All five MRSA isolates contained the mecC gene, were PVL negative, carried SCCmec type XI and belonged to ST130 (where ST stands for sequence type), with spa types t843, t10513 or t3256, or to ST2620, with spa type t4335. The MRnSA isolate, most closely related to Staphylococcus stepanovicii, carried mecA and blaZ genes related to SCCmec XI. MRSA isolates exhibited resistance to the β-lactams only. CONCLUSIONS The MRSA isolates described in this study represent the first detection of mecC-positive MRSA in a European otter (Lutra lutra) and a European brown hare (Lepus europaeus). The MRnSA isolate represents the first isolation of MRnSA from a Eurasian lynx (Lynx lynx).

Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells.

Mycoplasma (M.) bovis cytadhesion was studied using permanent embryonic bovine lung (EBL) cells as host system. Adherence rates were found to be strongly dependent on temperature and the mycoplasma-to-EBL ratio near the point of saturation of the attachment isotherm was determined to be 225 : 1. Mild trypsinization of viable M. bovis cells caused a measurable decrease of adherence indicating that surface proteins, among them the P26 antigen, played a major part as adhesion factors. Neuraminidase treatment of mycoplasmas led to a drastic reduction of adherence rates, which emphasizes the importance of sialyl moieties in adhesive interactions. The ability of the P26 antigen, a hydrophilic 32-kDa protein, to function as an adhesin was confirmed using a competitive adherence assay, in which the HPLC-purified protein was shown to reduce mycoplasma adhesion. These data complement previous findings obtained with the corresponding monoclonal antibody (MAb) 4F6. In further inhibition experiments, it could be demonstrated that MAb 1E5, which is directed against a common epitope of at least three members of the Vsp (variable surface protein) family of M. bovis, was also capable of decreasing mycoplasma attachment to EBL cells. This is the first evidence of possible involvement of Vsps in cytadhesion. In an effort to identify more putative adhesion proteins of this organism, the reverse adherence screening assay was used, a procedure based on the specific binding of labelled mammalian tissue culture cells to Western-blotted mycoplasmal proteins.

In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens

The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R/E were examined in western immunoblots. A reference anti-M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs 1E5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gallisepticum R or clonal variant R/E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA+p67a- phenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host environment at different stages of a natural infection.

Mycoplasma gallisepticum Invades Chicken Erythrocytes during Infection

ABSTRACT Recently, it was demonstrated using in vitro assays that the avian pathogen Mycoplasma gallisepticum is able to invade nonphagocytic cells. It was also shown that this mycoplasma can survive and multiply intracellularly for at least 48 h and that this cell invasion capacity contributes to the systemic spread of M. gallisepticum from the respiratory tract to the inner organs. Using the gentamicin invasion assay and a differential immunofluorescence technique combined with confocal laser scanning microscopy, we were able to demonstrate in in vitro experiments that M. gallisepticum is also capable of invading sheep and chicken erythrocytes. The frequencies of invasion of three well-defined M. gallisepticum strains were examined over a period of 24 h, and a significant increase in invasiveness occurred after 8 h of infection. In addition, blood samples derived from chickens experimentally infected via the aerosol route with the virulent strain M. gallisepticum Rlow were analyzed. Surprisingly, M. gallisepticum Rlow was detected in the bloodstream of infected chickens by nested PCR, as well as by differential immunofluorescence and interference contrast microscopy that showed that mycoplasmas were not only on the surface but also inside chicken erythrocytes. This finding provides novel insight into the pathomechanism of M. gallisepticum and may have implications for the development of preventive strategies.

Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host–pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site‐specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon‐based vector. Inactivation of xer1 abolished further Vpma switching and the ‘phase‐locked’ mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild‐type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other ‘difficult‐to‐manipulate’ mycoplasmas.