Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA)

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (K_d) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.     

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and WlBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.     

Biophysical and structural characterization of the small heat shock protein HspA from Thermosynechococcus vulcanus in 2 M urea

Small heat shock proteins (sHSPs) belong to the superfamily of molecular chaperones. They prevent aggregation of partially unfolded or misfolded client proteins, providing protection to organisms under stress conditions. Here, we report the biophysical and structural characterization of a small heat shock protein (HspA) from a thermophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 M urea. HspA has been shown to be important for the protection of Photosystem II and the Phycobilisome antenna complex at elevated temperatures. Heterologously expressed HspA requires the presence of 1–2 M urea to maintain its solubility at concentrations required for most characterization methods. Spectroscopic studies reveal the presence of the β-sheet structure and intactness of the tertiary fold in HspA. In vitro assays show that the HspA maintains chaperone-like activity in protecting soluble proteins from thermal aggregation. Chromatography and electron microscopy show that the HspA exists as a mixture of oligomeric forms in the presence of 2 M urea. HspA was successfully crystallized only in the presence of 2 M urea. The crystal structure of HspA shows urea-induced loss of about 30% of the secondary structure without major alteration in the tertiary structure of the protein. The electron density maps reveal changes in the hydrogen bonding network which we attribute to the presence of urea. The crystal structure of HspA demonstrates a mixture of both direct interactions between urea and protein functionalities and interactions between urea and the surrounding solvent that indirectly affect the protein, which are in accordance with previously published studies.     

HspA1A facilitates DNA repair in human bronchial epithelial cells exposed to Benzo[a]pyrene and interacts with casein kinase 2

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2 h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10 μM BPDE and p < 0.05 at 20 μM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10 μM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0454-7) contains supplementary material, which is available to authorized users.     

Immunoproteomics of extracellular proteins of Chinese virulent strains of Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is a porcine zoonotic pathogen with worldwide distribution, and lacking suitable vaccine and virulent maker were bottleneck to control this infection. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent Chinese SS2 strain ZY05719. The convalescent serum of a specific pathogen free (SPF) mini-pig recognized nine protein spots on PVDF membrane. Antigenic proteins on a duplicate gel, as well as those with a similar placement of extracellular proteins from another virulent strain (HA9801) and an avirulent strain (T15) on 2-D gels, were excised and identified by MALDI-TOF-MS. PMF of the protein spots were performed using the MASCOT server. Two proteins were found in all three strains. Comparative proteomic analysis between the two virulent strains and the avirulent strain revealed nine differential proteins, eight of which were successfully identified. Genes for six of the differentially expressed proteins were found in both virulent strains, and of those were present in the avirulent stain.     

MSG1, a surface-localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes

Mycoplasma suis is a member of the group of uncultivable haemoplasmas which colonise erythrocytes of a wide range of vertebrates. Adhesion to erythrocytes is the crucial step in the unique haemoplasma life cycle. Due to the lack of a cultivation system, no adhesion structures have been identified so far. In order to determine potential adhesion molecules of M. suis, we screened genomic M. suis libraries. The protein MSG1 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) similarity was identified. The encoding gene msg1 is 1011bp in size. The overall homology of the deduced amino acid sequence to GAPDHs of other pathogenic mycoplasmas ranged from 52.6% to 54.5%. Recombinant MSG1 expressed in Escherichia coli exhibited GAPDH activity. Immunoblot and immunoelectron microscopy analyses using antibodies against rMSG1 verified the membrane and surface localisation of native MSG1 in M. suis. Furthermore, we showed that rMSG1 binds to erythrocyte lysate in a dose-dependent manner. E. coli transformants which express MSG1 on their surface acquire the ability to adhere to porcine erythrocytes. This adhesion could be specifically and significantly inhibited by rMSG1 and antibodies to MSG1. In conclusion, our studies indicate that the membrane-associated MSG1 represents the first putative adhesion protein identified in the group of haemoplasmas.     

Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an alpha-crystallin-related, small heat shock protein.

A 16-kDa protein, one of the major proteins that accumulates upon heat-shock treatment in the thermophilic cyanobacterium Synechococcus vulcanus, was purified to apparent homogeneity. The N-terminal and internal amino acid sequences of the protein exhibited a homology to the alpha-crystallin-related, small heat shock proteins from other organisms. The protein was designated HspA. Size-exclusion chromatography and nondenaturing gel electrophoresis demonstrated that HspA formed a large homo-oligomer consisting of 24 subunits. It prevented the aggregation of porcine malic dehydrogenase at 45 degrees C and 50 degrees C and citrate synthase at 50 degrees C. The activity of the malic dehydrogenase, however, was not protected under these heat-shock conditions or reactivated after a shift in temperature from 45 or 50 degrees C to 21 degrees C. HspA was able to enhance the refolding of chemically denatured rabbit muscle lactate dehydrogenase in an ATP-independent manner. A homologue to the 16-kDa protein was also found to be induced upon heat-shock treatment in the mesophilic cyanobacterium Synechocystis sp. PCC 6803.     

First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples

Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies.     

A histidine-rich and cysteine-rich metal-binding domain at the C terminus of heat shock protein A from Helicobacter pylori: implication for nickel homeostasis and bismuth susceptibility

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.     

Use of western immunoblot analysis for testing moose serum for Brucella suis biovar 4 specific antibodies.

To determine if 12 moose (Alces alces) from northern Alaska with agglutinating antibodies specific for Brucella spp. had been exposed to either B. suis biovar 4 or B. abortus biovar 1, western immnnoblot serologic analysis was performed. Differential serologic responses to strain specific A and M antigenic variances of the lipopolysaccharide O-polysaccharide sugar allowed strain identification. Prior to examination, test sera were absorbed with killed whole cells from either B. abortus biovar 1, containing predominately A antigen (A+ M-); B. melitensis biovar 1, containing essentially M antigen (A- M+); or B. suis biovar 4, containing both antigenic tyes (A+ M+). The resulting sera were then examined by western immunoblot for recognition of either B. abortus biovar 1, B. melitensis biovar 1, or B. suis biovar 4 cell lysates. The results of this study indicate that these moose were exposed to B. suis biovar 4, a known pathogen of caribou (Rangifer tarandus) from arctic Alaska.     

Expression of UreB and HspA of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in silkworm pupae and identification of its immunogenicity

For mass production of urease B subunit (UreB) and heat shock protein A subunit (HspA) of Helicobacter pylori with Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) and to determine whether they could be used as an oral vaccine against H. pylori, besides, to determine the time course of expressed recombinant protein and the optimum acquisition time directly through green fluorescence, HspA and enhanced green fluorescence protein (EGFP) genes were cloned into vector pFastBacDual to form donor vector pFastBacDual-(EGFP) (HspA), UreB gene was cloned into vector pFastBacDual to form donor vector pFastBacDual-UreB,then they were transformed into E. coli BmDH10Bac to obtain the recombinant Bacmid-(EGFP) (HspA) and Bacmid-UreB respectively. They were used to transfect BmN cells and generated the recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB. Using these recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB inoculated the silkworm pupae, a recombinant HspA and UreB protein were expressed in silkworm pupae, which were around 13 and 62 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. After oral immunization of mice, serum specific IgG antibodies against HspA and UreB in vaccine group were much higher than that in mock and native silkworm powder control groups. The results indicated that the expressed recombinant HspA and UreB in silkworm pupae would possess good immunogenicity. In addition, when EGFP and HspA proteins were expressed, a direct correlation between the increase in intensity of fluorescence and HspA concentration.     

A small heat-shock protein confers stress tolerance and stabilizes thylakoid membrane proteins in cyanobacteria under oxidative stress

Small heat-shock proteins are molecular chaperones that bind and prevent aggregation of nonnative proteins. They also associate with membranes. In this study, we show that the small heat-shock protein HspA plays a protective role under oxidative stress in the cyanobacterium Synechococcus elongatus strain ECT16-1, which constitutively expresses HspA. Compared with the reference strain ECT, ECT16-1 showed much better growth and viability in the presence of hydrogen peroxide. Under the peroxide stress, pigments in thylakoid membrane, chlorophyll, carotenoids, and phycocyanins, were continuously reduced in ECT, but in ECT16-1 they decreased only during the first 24 h of stress; thereafter no further reduction was observed. For comparison, we analyzed a wild type and an hspA deletion strain from Synechocystis sp. PCC 6803 and found that lack of hspA significantly affected the viability of the cell and the pigment content in the presence of methyl viologen, suggesting that HspA stabilizes membrane proteins such as the photosystems and phycobilisomes from oxidative damage. In vitro pull down assays showed a direct interaction of HspA with components of phycobilisomes. These results show that HspA and small heat-shock proteins in general play an important role in the acclimation to oxidative stress in cyanobacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insights into the gene expression profile of uncultivable hemotrophic Mycoplasma suis during acute infection, obtained using proteome analysis

Hemotrophic mycoplasmas, bacteria without cell walls whose niche is the erythrocytes of their hosts, have never been cultivated in vitro. Therefore, knowledge of their pathogenesis is fundamental. Mycoplasma suis infects pigs, causing either acute fatal hemolytic anemia or chronic low-grade anemia, growth retardation, and immune suppression. Recently, the complete genomes of two hemotrophic mycoplasma species, M. suis and M. haemofelis, were sequenced, offering new strategies for the analysis of their pathogenesis. In this study we implemented a proteomic approach to identify M. suis proteins during acute infection by using tandem mass spectrometry. Twenty-two percent of the predicted proteins encoded in M. suis strain KI_3806 were identified. These included nearly all encoded proteins of glycolysis and nucleotide metabolism. The proteins for lipid metabolism, however, were underrepresented. A high proportion of the detected proteins are involved in information storage and processing (72.6%). In addition, several proteins of different functionalities, i.e., posttranslational modification, membrane genesis, signal transduction, intracellular trafficking, inorganic ion transport, and defense mechanisms, were identified. In its reduced genome, M. suis harbors 65.3% (strain Illinois) and 65.9% (strain KI_3806) of the genes encode hypothetical proteins. Of these, only 6.3% were identified at the proteome level. All proteins identified in this study are present in both M. suis strains and are encoded in more highly conserved regions of the genome sequence. In conclusion, our proteome approach is a further step toward the elucidation of the pathogenesis and life cycle of M. suis as well as the establishment of an in vitro cultivation system.     

Putative creatine kinase M-isoform in human sperm is identifiedas the 70-kilodalton heat shock protein HspA2

We previously described a putative creatine kinase M isoform in human sperm that is developmentally regulated and expressed during late spermiogenesis, simultaneous with cytoplasmic extrusion. We have now identified this protein as the testis-expressed 70-kDa heat shock protein chaperone known as HspA2 (the human homologue of mouse Hsp70-2). We have isolated and characterized HspA2 (formerly CK-M) by amino acid sequencing and have localized it by immunocytochemistry to spermatocytes at low levels, to spermatids, and in the tail of mature sperm. The specificity of the CK-M/HspA2 antiserum to HspA2 was demonstrated on immunoblots of one- and two-dimensional SDS-PAGE. In agreement with our earlier biochemical data, immunocytochemistry of testicular tissue indicated that HspA2 is selectively expressed in mature spermatids and in sperm about to be released in the seminiferous tubuli. The identity of HspA2 has been further confirmed by cross-absorption of the mouse HSP70-2 antibody by the HspA2/CK-M fraction, and by identical immunostaining patterns of human testicular tissue using either the anti-CK-M/HspA2 or an anti-mouse Hsp70-2 antisera. During spermiogenesis, both cytoplasmic extrusion and plasma membrane remodeling, which facilitate the formation of the zona pellucida binding site, involve major intrasperm protein transport, which may be chaperoned by HspA2. Accordingly, in immature human sperm, which fail to express HspA2, there is cytoplasmic retention and lack of zona pellucida binding. The present findings provide the biological rationale for the role of the human HspA2 as an objective biochemical marker of sperm function and male fertility, which we have established in earlier clinical studies.     

Significant contribution of the pgdA gene to the virulence of Streptococcus suis

Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S. suis peptidoglycan (PG) and found, among other modifications, N-deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N-deacetylation correlated with absence of significant lysozyme resistance when wild-type S. suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S. suis may enhance PG N-deacetylation under these conditions. Evaluation of the DeltapgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S. suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the DeltapgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N-deacetylation is an important factor in S. suis virulence.     

幽门螺杆菌热休克蛋白A基因的克隆、表达及免疫原性研究

幽门螺杆菌的感染可诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ（ＨｓｐＡ）可刺激机体产生保护性的免疫反应。用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序ＨｓｐＡ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＨｓｐＡ基因表达的蛋白质分子量约为１５ｋＤ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体。ＨｓｐＡ有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。     

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.     

Phenotypic changes of Helicobacter pylori components during an experimental infection in mice

The bacterial pathogen Helicobacter pylori is highly adapted to the human stomach and the clinical isolates show a high diversity which could be due to adaptative changes of the strains passing from one host to another. In order to study these variations, experimental infection of mice was developed and provided three out of the eleven tested strains able to infect C57BL/6 mice: the Sydney strain which is known to be well adapted to mice and two freshly isolated strains from infected patients. Mice were orally infected with one of these three strains (infecting strains) and were killed 45 days later. H. pylori strains were isolated from the stomachs of mice (emerging strains). The three infecting strains were compared to the three emerging strains for protein and lipopolysaccharide profiles, antigenic profiles revealed by Western blot with monospecific sera and genetic status by testing for the cagA gene and the vacA genotype. During the 45 days of infection, H. pylori underwent phenotypic variations which may be attributed to the adaptation from a human to a mouse environment or from an in vitro to a mouse environment. Those variations consisted of an over-expression at the cell surface of a 180-kDa protein and of a decreased expression of proteins of 260 and 120 kDa. Moreover, antigenic variations were shown for the two freshly isolated strains from human: the CagA and VacA antigens were in the saline extracts of the infecting strains only while the UreA, UreB, HspA and HspB were in the saline extracts of both the infecting and the emerging strains. These variations may contribute to the adaptation of the strains to the mouse environment.