Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.     

Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Group A streptococcus adheres to pharyngeal epithelial cells with salivary proline-rich proteins via GrpE chaperone protein

Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology.     

Serum opacity factor promotes group A streptococcal epithelial cell invasion and virulence

Serum opacity factor (SOF) is a bifunctional cell surface protein expressed by 40-50% of group A streptococcal (GAS) strains comprised of a C-terminal domain that binds fibronectin and an N-terminal domain that mediates opacification of mammalian sera. The sof gene was recently discovered to be cotranscribed in a two-gene operon with a gene encoding another fibronectin-binding protein, sfbX. We compared the ability of a SOF(+) wild-type serotype M49 GAS strain and isogenic mutants lacking SOF or SfbX to invade cultured HEp-2 human pharyngeal epithelial cells. Elimination of SOF led to a significant decrease in HEp-2 intracellular invasion while loss of SfbX had minimal effect. The hypoinvasive phenotype of the SOF(-) mutant could be restored upon complementation with the sof gene on a plasmid vector, and heterologous expression of sof49 in M1 GAS or Lactococcus lactis conferred marked increases in HEp-2 cell invasion. Studies using a mutant sof49 gene lacking the fibronectin-binding domain indicated that the N-terminal opacification domain of SOF contributes to HEp-2 invasion independent of the C-terminal fibronectin binding domain, findings corroborated by observations that a purified SOF N-terminal peptide could promote latex bead adherence to HEp-2 cells and inhibit GAS invasion of HEp-2 cells in a dose-dependent manner. Finally, the first in vivo studies to employ a single gene allelic replacement mutant of SOF demonstrate that this protein contributes to GAS virulence in a murine model of necrotizing skin infection.     

Fine surface structure of enterotoxemic Escherichia coli O139:K12 strains associated with swine edema disease

The fine structure of the cell surface of seven enterotoxemic Escherichia coli (ETEEC) O139:K12 strains isolated from piglets with edema disease were examined electron microscopically using both the negative-staining method and the freeze-substitution fixation method. Densely packed, fine fibers were observed; they consisted of a capsule layer approximately 25 nm thick around the cell surfaces of strains 107/86, IW-2, ED-3, ED-43, and ED-61, all of which have a capacity to adhere strongly to HEp-2 cells. In contrast, no such structure was observed on the surface of strains RK-O139 or ED-1, both of which adhere only weakly to HEp-2 cells. These results suggest that the capsule structure might be associated with the ability to adhere to HEp-2 cells and, as a result, also potentially play some role in ETEEC infection. Received: 29 April 1996 / Accepted: 13 August 1996     

Bacterial determinants of persistent throat colonization and the associated immune response in a primate model of human group A streptococcal pharyngeal infection

Group A streptococcal (GAS) pharyngitis and the subsequent bacterial colonization of the human throat elicit an immune response that may precipitate acute rheumatic fever in a susceptible host. To study the bacterial determinants that influence throat colonization and induction of humoral immunity, we characterized the behavior of GAS strains in a baboon model. An M-type 3 clinical isolate of GAS typical of strains that cause pharyngitis and invasive infection was recovered from the pharynx of six out of six baboons for at least 6 weeks after oral inoculation. By contrast, an isogenic mutant deficient in M protein failed to colonize most animals or was rapidly cleared. An isogenic mutant deficient in hyaluronic acid capsule colonized five out of six animals, but only persisted in the pharynx for 14–21 days. Colonized animals developed serum anti- streptolysin O (SLO) and anti-M protein immunoglobulin (Ig)G. The kinetics of the antibody responses were similar to those seen after human infection. Peak titres increased with the duration of throat carriage. Colonization with GAS prevented recurrent colonization after challenge with the homologous wild-type strain, but not after challenge with a strain of different M protein type. Early clearance of the M protein-deficient strain was associated with increased susceptibility of this strain to phagocytic killing in non-immune serum, whereas clearance of the acapsular strain was associated with increased susceptibility to phagocytic killing in the presence of specific antibody. These studies support critical and distinct effects of the GAS M protein and capsule on throat colonization and induction of humoral immunity in a model that reproduces important features of pharyngeal colonization and immune response following human infection.     

HEp-2 cell adherence and Vero cell cytotoxin production by EPEC strains isolated from children with diarrhoea in New Zealand

Abstract A total of 112 EPEC strains isolated from children with diarrhoea in New Zealand were examined for mannose-resistant HEp-2 cell adherence and production of exotoxins. Enterotoxin production was not detected in any of the strains examined. Verotoxin production was detected in 13 (11.6%) strains and of these 4 were also found to adhere to HEp-2 cells. HEp-2 cell adherence was displayed by a total of 29 (25.8%) strains of which 22 were diffusely adherent. Only 3 (2.7%) strains were shown to belong to the new virulence phenotype, entero-aggregative adherence, when examined in the adherence assay. We identified one strain with the novel characteristics of causing detachment of HEp-2 cells from glass coverslips and are further investigating this possible virulence mechanism. These results suggest that if EPEC strains are to be considered as a cause of diarrhoea, the search for new virulence factors must be extended.     

高毒力A组链球菌致病机制研究进展

A组链球菌(Group A Streptococcus,GAS)常导致咽炎和皮肤感染,也能引起严重侵袭性感染.根据其表面M蛋白编码基因emm可将GAS分为200多型,严重侵袭性感染多由高毒力株引起,以emm1、emm3、emm12、emm28和emm89型常见.研究发现高毒力GAS株中CovRS基因突变可导致细菌逃逸固...     

Isolation and characterization of cytotoxic, aggregative Citrobacter freundii

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 "cytotoxic and aggregative C. freundii." Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Differential binding to and biofilm formation on,HEp-2 cells by Salmonella enterica serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster

Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.     

An enteroaggregative Escherichia coli strain of serotype O111:H12 damages and invades cultured T84 cells and human colonic mucosa

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAEC) are not well defined. We investigated the interaction of EAEC strain 236 (serotype O111:H12) with polarised Caco-2 and T84 human intestinal epithelial cells lines, and with human jejunal and colonic mucosa. Strain 236 adhered to both polarised cell lines and to both intestinal tissue types, but caused severe damage and was invasive only in T84 cells and colonic mucosa. In contrast, prototype EAEC strain 042, which also adhered to the cultured intestinal cell lines, did not adhere to or invade jejunal or colonic tissue. These observations suggest a heterogeneity of virulence properties within the EAEC category of diarrhoea-causing E. coli.     

Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients

Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the C-terminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.     

Characterization of biofilm formation by clinically relevant serotypes of group A streptococci

Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

Non-Invasive Providencia alcalifaciens Strains Fail to Attach to HEp-2 Cells

We investigated the adherence properties of six P. alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells. Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection. In contrast, weakly or non-invasive P. alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection. Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E. coli (EPEC). These results suggest that the strain variation in the ability of P. alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates. Received: 10 April 2001 / Accepted: 17 April 2001     

Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.     

Adhesion of Lactobacillus acidophilus to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer)

Lactobacillus acidophilus was isolated from washed and homogenized walls of the crop and caecum of an adult fowl. A strain that adhered well in the Fuller adhesion test was subcultured until colonies on Lactobacillus Selective agar changed from rough to smooth. This coincided with a change from aggregate to planktonic growth in liquid medium and a marked loss of ability to adhere. The ultrastructure of cells from both types of culture was studied by electron microscopy. An S-layer formed the outermost part of the cell wall in the strongly-adherent strain, whereas this layer was covered with polymerized material or was absent in strains that lacked the ability to adhere, or those with reduced adherence.