Microbial Pathogenesis

Published by Elsevier
Online ISSN: 1096-1208
Publications
Article
In an attempt to study the effect of heterologous genes on the virulence of Vibrio cholerae 01 and non-01, rfb genes encoding biosynthesis of non-01 antigens were introduced by homologous recombination into the chromosome of V. cholerae 01 strain 569B (serotype Inaba, biotype classical). Recombinant strains were obtained which were not agglutinated with the diagnostic cholera 01 antiserum and were not sensitive to the cholera diagnostic bacteriophage, but produced as much cholera toxin as 569B and were highly virulent in the infant rabbit intraintestinal injection model. These data indicate that the rfb genes from the studied V. cholerae non-01 did not alter the virulence phenotype of V. cholerae 01. In contrast, cloned ctxAB genes from V. cholerae 01 encoding cholera toxin introduced into a non-pathogenic strain lead to efficient secretion of cholera toxin but to only low virulence in the infant rabbit model.
 
Article
The genes responsible for the biosynthesis of the O1 polysaccharide from a human pyelonephritic Escherichia coli were cloned and expressed in a rfb-deleted E. coli K-12 strain. Deletion analysis of the clone demonstrated that a DNA fragment size larger than 6.7 kb and smaller than 10 kb is responsible for O1-antigen biosynthesis.
 
Article
Enteropathogenic Escherichia coli (EPEC) can adhere to, invade and multiply in human epithelial cells. To define the elements required for bacterial invasion, we isolated from an 0111:H- EPEC a 6.6 kb plasmid that is capable of conferring to an avirulent, non-adherent E. coli K12 strain (DK1) the capacity to invade epithelial cells. With this system a dissociation was possible between bacterial invasion and adherence to epithelial cells. Bacteria containing this plasmid synthesise a protein of 32 kDa (pl 4.93) which seemed to be required for cell invasion. The results provide a new basis for strategies to prevent EPEC infections.
 
Article
Mutants of Vibrio cholerae 01 strain 0395 (classical) mutated in genes encoding secretory or cell surface proteins were induced by TnphoA mutagenesis and were selected as blue colonies on L-agar plates containing 5-bromo-4-chloro-3-indolyl phosphate. Southern analysis of the total DNA from blue colonies showed that all mutants had TnphoA insertion in genomic DNA. These mutants were analysed for adherence, colonization and protein profile. Adherence to freshly isolated rabbit intestinal discs was affected in some mutants. The less adhesive mutants were examined for colonization of the intestine of infant mice. One mutant, designated T-87, was extremely poor at colonization and less diarrhaegenic than the parent strain. Analysis of T-87 by SDS-PAGE revealed that two proteins of 53 and 38 kDa were lacking. The 38 kDa protein was identified as OmpU. The 53 kDa protein was extracellular and cells treated with anti-53-kDa antibodies could not colonize the gut of infant mice. The expression of the 53 and 38 kDa proteins in T-87 was dependent of the growth medium. The data suggest that T-87 is mutated in a regulatory gene which regulates the expression of proteins involved in intestinal colonization.
 
Article
The nucleotide sequence of a trans-acting P-fimbrial regulatory element obtained from the uropathogenic Escherichia coli strain KS71 (04:K12) was determined. The regulatory element was found to contain an open reading frame of 231 nucleotide residues that showed 95.2% homology with papl, a functionally analogous regulatory gene of E. coli strain J 96.
 
Article
High-affinity binding sites for P-fimbriated and for 075X-positive Escherichia coli were located in the human kidney. Frozen sections of normal human kidney were double-stained first with fluorochrome-labelled bacteria and then with fluorochrome-labelled nephron site-specific lectins or antibodies. The P-fimbriate recombinant E. coli strain used showed specific adherence to glomerular structures, to the lumen of proximal and sital tubules and to vascular endothelium but did not adhere to collecting ducts or to peritubular sites. Two E. coli strains having the 075X adhesin showed specific adherence to renal interstitium, to glomerular elements and to Bowman's capsule. The method described allows the detailed determination of tissue-substructure specificity of bacterial adhesion. Our results demonstrate tissue tropism in the adhesion of E. coli to human kidneys and suggest a pathogenetic role for X adhesins.
 
Article
Shiga toxin-producing Escherichia coli (STEC) 091:H21 strain B2F1, an isolate from a patient with the hemolytic uremic syndrome (HUS), produces elastase-activatable Shiga toxin (Stx) type 2d and adheres well to human colonic epithelial T84 cells. This adherence phenotype occurs even though B2F1 does not contain the locus of enterocyte effacement (LEE) that encodes the primary adhesin for E. coli O157:H7. To attempt to identify genes involved in binding of B2F1 to T84 cells a bank of mini-Tn5phoACm(r) transposon mutants of this strain was generated. Several of these mutants exhibited a reduced adherence phenotype, but none of the insertions in these mutants were within putative adhesin genes. Rather, insertional mutations within hns resulted in the loss of adherence. Moreover, the hns mutant also displayed an increase in the production of hemolysin and alkaline phosphatase and a loss of motility with no change in Stx2d-activatable expression levels. When B2F1 was cured of the large plasmid that encodes the hemolysin, the resulting strain adhered well to T84 cells. However, an hns mutant of the plasmid-cured B2F1 strain exhibited a reduction in adherence to T84 cells. Taken together, these results indicate that H-NS regulates the expression of several genes and some potential virulence factors in the intimin-negative B2F1 STEC strain and that the large plasmid is not required for T84 cell colonization.
 
Article
The toxigenic element of Clostridium difficile strain VPI 10463 is identified by establishing boundaries between toxigenic sequences and those sequences shared by nontoxigenic and toxigenic strains. The toxigenic element is chromosomal, 19.6 kb in length, and comprised of five open reading frames which include the toxin A and B genes. Four of the open reading frames are contiguous and are transcribed in the same direction. The fifth is downstream from the others and oriented in the opposite direction. One of the open reading frames, located 5' to the toxin B gene, is previously unknown. Both upstream (5') and downstream (3') boundaries for the toxigenic element were examined in six toxigenic strains which vary considerably in toxigenicity to determine if there were variations among their respective toxigenic elements. The toxigenic element is highly conserved in these six strains. In the three nontoxigenic strains examined, a short fragment (127 bp) occupies the same chromosomal location as the large 19.6 kb toxigenic element.
 
Article
While there is a growing consensus on the understanding that the immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system, little is known about the key players in the first steps of the infection and about the sites of the disease development. Owing to their subepithelial location and their migratory capacity, macrophages could be early targets for prion transportation or propagation during the later stages of disease. In order to investigate the role of macrophages, we studied in vitro the effect of exposing primary peritoneal macrophages to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP 106-126. As shown by MTT assay, macrophage viability treated with less than 50 microM PrP 106-126 for 72 h was not inhibited but slightly stimulated at 10 and 25 microM, while there was significant decrease when exposed to 100 microM PrP 106-126 for 72 h. The expressions of PrP at mRNA and protein level were up-regulated following treatment with PrP 106-126 for 72 h. Cytokine TNF-alpha production were elevated by the PrP peptide in a time-dependent manner, which demonstrated a proinflammatory response linked to the presence and progression of prion disease took place in macrophages. These findings suggested that macrophages may play roles in the transportation and replication of the infectious agent.
 
Article
Infection of susceptible weaned pigs with oedema disease strains of E. coli is associated with bacterial adhesion to the small intestine. F18 fimbria (previously named F107) was the first colonisation factor described on oedema disease strains, and its genetic determinant was cloned. In the present study, genes fedE and fedF were positioned in the F18 gene cluster, downstream of the major structural subunit gene fedA. Two fedE and two fedF mutants were identified that had lost their capacity to adhere to isolated porcine villi. Moreover, these mutants produced significantly longer fimbriae. In vitro adhesion tests, electron microscopy study, transcomplementation tests, and nucleotide sequence analysis indicated that proteins FedE and FedF are F18 minor subunits essential for fimbrial adhesion and effecting fimbrial length.
 
Article
The impact of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine alveolar macrophages (Mo) was examined by differential display reverse transcription PCR (DDRT-PCR). A PRRSV-induced expressed gene tag (EST) was used to isolate and identify a single cDNA clone from a library prepared from porcine peripheral blood. Rapid amplification of cDNA ends (RACE) was employed to clone a 1.5 kb fragment at the 5' end of the mRNA. DNA sequencing identified an open reading frame (ORF) of 2820 bp. Deduced amino acid sequence revealed the eight conserved domains characteristic of the DEAD/H box protein superfamily. The putative porcine RNA helicase induced by virus (RHIV -1) showed 84% amino acid similarity to human retinoic acid-induced gene (RIG-I). Porcine RHIV -1 transcripts were ubiquitously expressed in various pig tissues, while in PRRSV-infected pigs, higher expression was observed in several tissues persistent for PRRSV. These data indicate the association of PRRSV genome replication with enhaced host cell RNA helicase gene expression. Finally, the RHIV -1 gene was localized on porcine chromosome 10q13 between markers SSC25A02 and SWR334 via somatic cell panel and radiation hybrid (RH) mapping strategies.
 
Article
This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.
 
Article
A number of different techniques were used to analyse classic and atypical serogroup O:11 Aeromonas isolates. Five of seven atypical O:11, S layer-negative strains lacking a homogeneous LPS side-chain pattern exhibited varying degrees of mouse pathogenecity. One virulent atypical strain (AH-77) synthesized a surface array protein (SAP) but was unable to anchor it to the cell surface in an intact form, presumably due to a defect in its LPS architecture. Proteinase K digestion to remove the SAP, or growth at elevated temperatures (42 degrees C) to reduce the proportion of SAP synthesized from classic O:11 S layer-positive strains, did not alter their LD50 values in outbred mice. In addition, a spontaneous mutant, AS-180-1, that was S layer-negative was as virulent as the parental S layer-positive strain in the mouse model. These results suggest neither the SAP nor the characteristic serogroup O:11 homogeneous LPS side-chain pattern are directly involved in mouse pathogenicity.
 
Article
Since it has been reported that a single amino acid mutation of Gly-->Arg in the CAGYC region of the beta chain of human thyroid stimulating hormone (hTSH) was responsible for congenital isolated TSH deficiency, and that the same amino acid substitution in this site of hTSH and human chorionic gonadotropin (hCG) introduced by site-directed mutagenesis resulted in loss of activity, the authors studied the role of glutamic acid at position 11 (Glu-11) from the N-terminus of the B subunit of cholera toxin (CT), which corresponds to the glycine in the CAGYC region of the beta chain of hTSH and hCG. A mutant CT constructed by site-directed mutagenesis in which Glu-11 was replaced by Arg (CT-E11R) did not induce either morphological changes or accumulation of cytosolic cyclic AMP in Chinese hamster ovary cells, although it formed the holotoxin AB5, retained the ability to bind to GM1-ganglioside and showed ADP-ribosyltransferase activity. Weak assembly of the B subunits in mutant CT-E11R demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-heating conditions might explain the loss of biological activity.
 
Article
To determine the role of hemolysin(s) in virulence and immunoprotection, non-hemolytic mutants of Actinobacillus pleuropneumoniae serotype 5, strain J45, were isolated following chemical mutagenesis. One mutant was selected for extensive characterization. Differences in capsule content, or in lipopolysaccharide or membrane protein electrophoretic profiles of the parent and mutant were not detected. A predominant, calcium-inducible protein of 110 kDa was present in culture supernatant of the parent, but absent from the mutant. Two-dimensional (2-D) gel electrophoresis confirmed that the 110 kDa protein was absent in culture supernatant of the mutant, but few, if any, minor differences could be detected in whole-cell proteins between the parent and mutant. The mutant totally lacked extracellular hemolytic and cytotoxic activity. Lysates of whole cells of the mutant contained weak hemolytic activity, and the 110 kDa protein could be detected by immunoblotting. Neutralization titers were negative in pigs immunized with the mutant or purified, denatured hemolysin, although enzyme-immunoassay titers were detected. Four additional independently isolated non-hemolytic mutants were avirulent in pigs and mice at doses greater than 10 times the lethal dose of the parent. Neither pigs nor mice were protected against lethal infection following immunization with the non-hemolytic mutant. We conclude that the 110 kDa hemolysin plays an important role in bacterial virulence and the pathogenesis of pleuropneumonia, and that sufficiently high levels of neutralizing antibodies to the 110 kDa hemolysin may be required for protection of pigs against disease.
 
Article
It has previously been reported that the relative rate of synthesis of K88 fimbriae was influenced by the growth phase of Escherichia coli K88. Consequently, the effect of growth phase on the adhesive capacity of the K88 bearing cells of E. coli strain Bd 1107/7508 (K88ac) has now been investigated. The adhesion process was studied in terms of adhesion to and affinity for the immobilized piglet ileal mucus, the latter by applying Michaelis-Menten kinetics. The amount of K88 fimbriae per cell was determined by both the ELISA technique and by quantitative 1D-gel electrophoresis. The adhesion to, as well as the affinity for the mucus receptors was shown to be rather constant throughout growth. In agreement with these findings the cells collected at the different growth phases were found to be equally fimbriated. From these results it is concluded that, although the relative synthesis rate of K88 fimbriae varied during the growth cycle, the amount of K88 fimbriae expressed on the E. coli cell surface and the adhesive capacity of E. coli strain Bd 1107/7508 is largely constant throughout growth.
 
Article
A DNA sequence, homologous to the cfaD gene of CFA/I region 2, was identified on CFA/I region 1. This sequence is designated cfaD'. It differs from the cfaD gene in containing two deletions and a stop codon. The cfaD' sequence therefore can only encode a truncated CfaD-like protein. The CfaD protein may be a DNA binding protein and functions as a positive regulator of CFA/I fimbriae expression. A regulatory function for the cfaD' is not likely since deletion of the cfaD' sequence does not affect production of CFA/I fimbriae in E. coli K-12 strains. That the cfaD' sequence is present on CFA/I wild-type plasmids isolated from CFA/I strains of different serotypes, obtained at various geographical locations, suggests, however, that this DNA region is not completely without a function.
 
Article
The immunodominant 120 kDa protein (p120) of Ehrlichia chaffeensis was demonstrated to be exposed on the surface of purified whole ehrlichial cells examined by immunoelectron microscopy with a rabbit antibody against a portion of the domain containing tandem repeat units. In the intracellular location, the 120 kDa protein was detected by immunoelectron microscopy in the outer membrane of the cell wall of dense-core forms of the ehrlichiae in infected canine macrophage-like cells and as a component of the intramorular fibrillary matrix. No 120 kDa protein was detected in the cell wall of ehrlichial reticulate cells. Recombinant Escherichia coli with a plasmid containing the entire 120 kDa protein gene, but no bacteria with non-recombinant plasmid, attached to the surface of HeLa cells as visualized by electron microscopy. Some of the recombinant 120 kDa protein expressing E. coli invaded the HeLa cells as determined by gentamicin protection assays and by intravacuolar localization ultrastructurally.
 
Article
In this study we compared the host response to Listeria monocytogenes in 129 REJ mice with listeria-resistant (C57Bl/6j) and susceptible (Balb/c) mouse strains. In all experiments mice were inoculated by the i.p. route. 129 REJ mice and Balb/c mice were sensitive to listeriosis whilst C57Bl/6j mice were relatively resistant to i.p. infection. Relatively large numbers of viable bacteria could be detected in the spleens of 129 REJ mice as early as 6 h following i.p. inoculation suggesting that dissemination of listeria from the peritoneal cavity is rapid in this mouse strain. This contrasted with Balb/c mice which exhibited an early lag phase during which only low numbers of bacteria could be isolated from the spleens of infected animals. In response to both proteose peptone and live listeria, 129 REJ mice demonstrated a greater capacity to recruit neutrophils to the peritoneal cavity than Balb/c and C57Bl/6j mice. In addition, inflammatory phagocytes from 129 REJ mice were as bactericidal in vitro as phagocytes from the Balb/c and C57Bl/6j strains. However, in vivo, inflammatory neutrophils elicited by proteose peptone prior to i.p. infection with L. monocytogenes were not protective in the three mouse strains tested. Despite the apparent inadequacy of peritoneal neutrophils in controlling early bacterial proliferation, depletion of neutrophils in 129 REJ mice severely exacerbated i.p. infection with L. monocytogenes. The results indicate that neutrophils provide an inefficient but essential means of controlling early outgrowth of listeria in the peritoneal cavity of 129 REJ mice. The excessive inflammatory response seen in 129 REJ mice may facilitate the early dissemination of L. monocytogenes from the peritoneal cavity to peripheral sites.
 
Article
A quantitative and kinetic study of the release of the hematopoietic cytokines IL-3, IL-5 and GM-CSF, the immunoregulatory cytokine IL-12 heterodimer (and its p40 subunit) and IL-13 by human peripheral blood mononuclear cells (PBMC) stimulated in vitro with the superantigen streptococcal pyrogenic (erythrogenic) exotoxin A (SPE A) from Streptococcus pyogenes is reported. PBMC were stimulated in parallel with heat-killed group A streptococcal cells, E. coli lipopolysaccharide (LPS) and with concanavalin A (Con A) in certain experiments for comparative purposes. The cytokines were assayed in the supernatant fluids by ELISA. IL-13 expression was also determined by a quantitative competitive PCR. IL-3, IL-5, GM-CSF, IL-12 p40, IL-12 heterodimer and IL-13 expression was induced by SPE A in a time- and dose-dependent manner in rather substantial amounts except the IL-12 heterodimer, which was released in small quantities. In contrast to SPE A, IL-3, IL-5 and IL-13 were not or poorly elicited by streptococcal cells or LPS whereas these two stimulants induced relatively high amounts of GM-CSF. Interestingly, both IL-12 p40 and IL-12 heterodimer were released in much higher amounts by streptococcal cells. Con A induced IL-3, IL-5, GM-CSF and IL-13 production in amounts comparable to those elicited by SPE A. The possible pathophysiological relevance of the elicitation by SPE A and streptococcal cells of these cytokines is discussed.
 
Results of PCR analysis of 24 PEPEC O45 LEE-positive isolates to distinguish an intact selC locus from one disrupted by insertion of the LEE
Article
In the present study, attaching and effacing Escherichia coli (AEEC) O45 isolates from post-weaning pigs with diarrhoea were examined for the presence of the LEE (locus of enterocyte effacement) using various DNA probes derived from the LEE of human enteropathogenic E. coli (EPEC) strain E2348/69. The LEE fragment was conserved among the eae -positive pig isolates. The attaching and effacing activity of PEPEC (pig EPEC) O45 isolates is highly correlated with the presence of the LEE. Nevertheless, for some PEPEC isolates, the insertion site of the LEE is different or has diverged during evolution. The presence of the LEE fragment in PEPEC isolates provides further evidence that the LEE region is conserved among AEEC of different animal origins. In addition, the nucleotide sequence of the region containing the eae gene and esp genes of a pig AEEC isolate, strain 1390, was determined. Among examined Eae proteins, Eae of strain 1390 showed the highest similarity with Eae belonging to the beta intimin group such as the Eae of rabbit AEEC. Moreover, all pig strains that produced attaching and effacing lesions in piglets and pig ileal explants belonged to the beta intimin group. The deduced amino acid sequences of the EspA, EspB and EspD proteins of strain 1390 showed particularly strong homology to those of AEEC strains presenting a beta intimin allele. Thus, pig AEEC possess the LEE sequences, and for the strain 1390, sequences of the eae and esp regions are related to those of other AEEC, in particular, strains presenting a beta intimin allele, such as the rabbit AEEC.
 
Article
In this study, we first assessed the effect of intragastric infection of pregnant mice with Listeria monocytogenes on relative expression of select genes associated with T cell subsets. Relative gene expression was moderately increased in placental tissues for IFNγ, IL-4, IL-17a, IL-22, CD3, and FoxP3. To assess the roles of IL-17a and IL-22 in resistance to listeriosis during pregnancy, we compared the severity of maternal and fetal infection in IL-17a((-/-)), IL-22((-/-)), and IL-17a((-/-))/IL-22((-/-)) mice with that of wild type C57BL/6 mice. Intragastric infection with modest numbers of bacterial cells (10(5) CFU) caused reproducible maternal and fetal infection in all four mouse strains. We recovered greater numbers of CFU from the bloodstream of pregnant IL-22((-/-)) mice than pregnant wild type mice. Otherwise we found no significant difference in bacterial load in maternal or fetal tissues (spleen, liver, fetoplacental units) from pregnant IL-17a((-/-)), IL-22((-/-)), or IL-17a((-/-))/IL-22((-/-)) or wild type mice. Nor did we observe histopathologic differences in severity of inflammation in maternal or fetal tissues from the various groups of mice. Although IL-17a and IL-22 are up-regulated in placental tissue, our study suggests that antibacterial resistance and the host inflammatory response are not dependent on IL-17a or IL-22 during infection of mice with L. monocytogenes at 10-14 days of gestation.
 
Article
Three clones from a strain of Salmonella choleraesuis (serogroup C1) were lysogenized with phage 14 (P14) which converts the O-antigen of serogroup C1 salmonellae from O-6,7 to O-6,7,14. The lysogens were compared with their parental non-lysogenic clones with respect to the following properties: average length of O-antigen polysaccharide chains, sensitivity to normal human serum, and mouse-virulence. SDS-polyacrylamide gel electrophoresis of lipopolysaccharides extracted from these bacteria showed that samples from lysogens consisted mainly of long-chained molecules whereas those from non-lysogens contained mainly short-chained molecules. The O-antigen polysaccharide from a lysogen was estimated by chemical analysis to be six times as long as that from a non-lysogen. Lysogens were serum-resistant whereas non-lysogens were serum-sensitive. About 10 times more colony forming units of a lysogen than of a non-lysogen were recovered from the livers and spleens of mice on day 1 and 3 after intraperitoneal inoculation of equal doses. By comparison with S. choleraesuis, lysogenization of S. typhimurium with phage P22 or phage A4 did not affect the chain-length distribution of O-antigen polysaccharide. Our data suggest that phage 14-coded determinants increase efficiency of O-antigen biosynthesis in S. choleraesuis leading to increase in average length of O-polysaccharide chains. Increased serum resistance and mouse virulence are logical consequences of increase in average length of O-polysaccharide chains and represent phage-conferred selective advantage not previously described in Salmonella.
 
Article
Tissue susceptibility and resistance to infection with the yeast Candida albicans is genetically regulated. Analysis of the strain distribution pattern of the C. albicans resistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant for Carg1, Tcra and Rib1. Therefore, Carg1 is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes.
 
Article
Tannerella forsythia is a Gram-negative oral anaerobe implicated in the development of periodontitis, a chronic inflammatory disease induced by bacterial infections which leads to tooth loss if untreated. Since biofilms formed by periodontal bacteria are considered important in disease progression and pose difficulties in treatment, we sought to investigate the underlying mechanisms of T. forsythia biofilm formation. This was carried out by screening random insertion mutants of T. forsythia for alterations in biofilm development. This approach lead to the identification of an operon involved in exopolysaccharide (EPS) synthesis. An isogenic mutant of one of the genes, wecC, contained within the operon was constructed. The isogenic wecC mutant showed increased ability to form biofilms as compared to the parent strain. The wecC mutant also formed aggregated microcolonies and showed increased cell-surface associated hydrophobicity as compared to the parent strain. Moreover, biochemical characterization of the wecC mutant indicated that glycosylation of surface glycoproteins was reduced. Therefore, our results suggest that the wecC operon is associated with glycosylation of surface-glycoprotein expression and likely plays an inhibitory role in T. forsythia biofilm formation.
 
Article
Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-gamma and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8+ T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.
 
Article
Mycobacterium paratuberculosis (MPT), the agent of paratuberculosis is a slow growing mycobacteria that causes important economic losses mainly due to lower weight gains and drastic decrease in milk production. Existing paratuberculosis vaccines are not completely protective and induce antibodies/delayed type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are needed to improve the control of this disease. We have identified, expressed and purified a hypothetical thiol peroxidase of MPT (MPT-TP) in mice. We also characterized the immunogenicity of this antigen in mice. The recombinant MPT-TP (rMPT-TP) antigen induced a high production of IFNgamma, IL-6, and NO and a low production of IL-10 by spleen cells of immunized mice. Addition of Ribi adjuvant to rMPT-TP resulted in lower IFNgamma secretion and higher NO production in spleen cells. A similar level of proliferation of spleen cells exposed to rMPT-TP was found in immunized groups (rMPT-TP and rMPT-TP emulsified in Ribi). DTH responses in mice footpads were observed only in mice immunized with rMPT-TP emulsified in Ribi. Addition of Ribi adjuvant clearly induced a significantly higher anti-rMPT-TP antibody production of all classes tested and decreased the IgG1/IgG2a ratio. MPT-TP demonstrated antigenic characteristics that make this antigen a potential component in the development of a future subunit vaccine against paratuberculosis.
 
Article
It has been previously shown that a surface complement regulatory protein (CRP) of Trypanosoma cruzi trypomastigotes binds human complement components C3b and C4b, and inhibits C3 convertase formation, thus contributing to the resistance of the bloodstage parasites to complement-mediated lysis. The blood stage parasites rapidly and spontaneously release a limited set of membrane glycoproteins, including CRP, and the modulation of release of CRP following ligand binding was investigated. Incubation of the parasites with C3b results in the release of CRP at a reduced apparent molecular mass. To determine if proteolytic processing was responsible for the reduction in apparent molecular mass of the released CRP, the proteolytic activity present in trypomastigote membrane preparations was examined. In addition to a well described cysteine protease, a novel 75 kDa protease was identified in tissue culture-derived trypomastigotes and axenically-derived metacyclic trypomastigotes membrane preparations. This protease was inhibited by aprotinin and leupeptin, but not L-trans-epoxysuccinyl-leucylamide-(4-guanidinobutane), N-[N-L-3-transcarboxyirane-2-carbonyl-L-leucinyl]-agmatine (E64). Treatment of the parasites with protease inhibitors did not affect spontaneous shedding of proteins, however, protease inhibitors abrogated the effect of C3b-binding on CRP degradation. These results indicate that binding of C3b to the CRP renders the CRP susceptible to cleavage by a parasite protease, possibly as a means of removing the CRP-C3b from the parasite surface. This process may represent an additional immune evasion mechanism which allows the parasite to avoid both complement-mediated lysis and clearance.
 
Article
Brucella is a broad-range, facultative intracellular pathogen that can survive and replicate in an endoplasmic reticulum (ER)-derived replication niche by preventing fusion of its membrane-bound compartment with late endosomes and lysosomes. This vacuolar hijacking was demonstrated to be dependent on the type IV secretion system VirB but no secreted effectors have been identified yet. A virB mutant is unable to reach its ER-derived replicative niche and does not multiply intracellularly. In this paper, we showed that, by co-infecting bovine macrophages or HeLa cells with the wild type (WT) strain of Brucella melitensis 16M and a deletion mutant of the complete virB operon, the replication of DeltavirB is rescued in almost 20% of the co-infected cells. Furthermore, we demonstrated that co-infections with the WT strains of Brucella abortus or Brucella suis were equally able to rescue the replication of the B. melitensis DeltavirB mutant. By contrast, no rescue was observed when the WT strain was given 1h before or after the infection with the DeltavirB mutant. Finally, vacuoles containing the rescued DeltavirB mutant were shown to exclude the LAMP-1 marker in a way similar to the WT containing vacuoles.
 
Article
A T-cell-stimulating 17 kDa protein of the vaccine strain Francisella tularensis LVS has previously been cloned, sequenced and shown to be a lipoprotein. In the present study, it was investigated whether the protein, denoted TUL4, and its gene are present in various strains of the genus Francisella. By Western blot analysis, it was demonstrated that a TUL4-specific monoclonal antibody bound to a protein present in each of the Francisella strains. The immunoreactive proteins had an M(r) of 17 kDa in all F. tularensis strains and in the strain Francisella novicida, whereas the M(r) in strains of Francisella philomiragia was 20 kDa. When genomic preparations were probed with a radioactive DNA fragment of F. tularensis LVS encoding TUL4, hybridization was demonstrated in all strains of Francisella, although the F. philomiragia strains did not hybridize under conditions of high stringency. The hybridizing chromosomal DNA fragment of the F. philomiragia strains was larger than that of the other Francisella strains. No hybridization or Western blot reactivity was seen when various other Gram-negative and Gram-positive bacteria were probed. In summary, the 17 kDa lipoprotein of F. tularensis LVS appears to be Francisella-specific and present in the species F. tularensis and F. novicida, whereas an immunologically related protein is present in F. philomiragia.
 
Article
Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization. Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments. Five different epitopes were identified that are involved in neutralization of viral infectivity. Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known. The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed.
 
Article
We have recently identified two markers associated with virulent strains of bluetongue virus serotype 17. These differences are an altered antigenic structure of the outer capsid protein VP2 and an increased electrophoretic mobility of the RNA segment 3 that codes for an inner core protein. We did not observe these markers in confirmed avirulent strains of bluetongue virus serotype 17. We hypothesized that these virulence-associated markers may have been acquired by bluetongue-17 through genetic interaction with other circulating serotypes of the virus. To test this hypothesis, we studied all isolates of other BLU serotypes obtained from the same sentinel cattle herds in Central America and the Caribbean on the same days as BLU-17 isolates. We looked for evidence of common epitopes on VP2 or an RNA segment 3 of identical mobility to that of the virulent strains of BLU-17. We found no evidence to indicate that genetic interaction with other co-circulating serotypes gave rise to these two specific virulence-associated markers of BLU-17.
 
Article
Comparison of the primary structures of the A subunits of Vero toxin 1 (VT1), Vero toxin 2 (VT2), and two variants of VT2 (VT2vp and VT2vh) and the ricin A chain revealed three conserved regions (amino acid residues 51-55, 167-171 and 202-207 from the N-terminus of VT1). All three regions of the ricin A chain corresponded in position to the active site of ricin proposed by X-ray crystal diffraction analysis. To determine the relative importance of the conserved amino acid residues for toxin activity of VT1, we prepared VT1 mutants with single amino-acid substitutions by oligonucleotide-directed site-specific mutagenesis. A total of 22 mutants were prepared to examine 14 conserved residues, and their cytotoxicities to Vero cells and inhibitory activities on protein synthesis in a rabbit reticulocyte lysate were compared with those of wild-type VT1. Replacement of glutamic acid at position 167 by glutamine and of arginine at position 170 by leucine reduced both activities drastically. These results suggest that, in addition to the glutamic acid at position 167 reported previously, arginine at position 170 also plays an important role in the toxin activity of VT1. A possible chemical mechanism of the enzymatic (N-glycosidase) activity of VT1 is proposed based on the relative activities of various mutants.
 
Article
Although proteases are recognized as important virulent factors in pathogenic microorganisms, little information is available so far regarding the potential role of these enzymes in diseases caused by mycobacteria. Here we use bioinformatic tools to compare the protease-coding genes present in the genome of Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis. This analysis allowed a review of the nomenclature of the protease family present in mycobacteria. A special attention was devoted to the 'decaying genome' of M. leprae where a relatively high level of conservation of protease-coding genes was observed when compared to other genes families. A total of 39 genes out of the 49 found in M. bovis were identified in M. leprae. Of relevance, a core of well-conserved 38 protease genes shared by the four species was defined. This set of proteases is probably essential for survival in the host and disease outcome and may constitute novel targets for drug development leading to a more effective control of mycobacterial diseases.
 
Article
The subcloning in pBR322 of the gene of the S. mutans OMZ 175 74K SR protein, was performed after in vitro reconstitution, from two recombinant EMBL3 phages, lambda SmAD9 and lambda SmAD10. The gene is expressed in E. coli HB101 under the control of its own promoter and produces a protein with a molecular weight of 195 kDa. A strong immunological relationship between the expressed protein and the 74K SR protein was observed in ELISA, Western blotting and immunoprecipitation. The 195 kDa protein was purified by immunoaffinity chromatography to homogeneity as judged by SDS-PAGE and native PAGE. Its reactivity with monoclonal anti 74K SR antibodies indicates that it is probably a precursor form of the 74K SR protein produced in S. mutans. The adhesion properties of the two proteins, tested in solid phase adherence assays, are quite similar. This indicates that the additional peptide present in the precursor protein has little or no role in the adherence properties of protein 74K SR.
 
Article
Analyses of invasive enteric bacteria (e.g. Shigella, Salmonella, Listeria, and Campylobacter) have shown that these pathogens initiate orchestrated signal transduction cascades in host cells leading to host cytoskeletal rearrangements that result in bacterial uptake. This current study was specifically aimed at examining the involvement of host membrane caveolae and certain protein kinases in epithelial cell invasion by C. jejuni strain 81-176, for which we have previously characterized the kinetics of entry and a unique microtubule-dependent mechanism of internalization. Utilizing in vitro cultured cell invasion assays with a gentamicin-kill step, disruption of membrane caveolae by pretreatment of INT407 cell monolayers with filipin III reduced C. jejuni 81-176 entry by >95%. Strain 81-176 uptake into INT407 cells was markedly inhibited by monolayer pretreatment with the protein kinase inhibitors genistein and staurosporine, or specific inhibitors of PI 3-kinase, wortmannin and LY294002. Western blot analysis using monoclonal anti-protein tyrosine phosphorylation antibody revealed distinctive changes during invasion in phosphorylation of at least nine proteins. Further inhibitor studies indicated that heterotrimeric G proteins, plus ERK and p38 MAP kinase activation are also involved in C. jejuni 81-176 invasion. These results suggest that C. jejuni 81-176 interact at host cell surface membrane caveolae with G protein-coupled receptors, which presumably trigger G-proteins and kinases to activate host proteins including PI 3-kinase and MAP kinases, that appear to be intimately involved in the events controlling 81-176 internalization.
 
Article
Escherichia coli F-18, a human fecal isolate, makes type 1 fimbriae in vitro and in the streptomycin-treated mouse large intestine in vivo, and is an excellent colonizer of the cecal mucus layer in the streptomycin-treated mouse large intestine. E. coli F-18(pPKL91) harbors an extra fimB gene on a parB stabilized pPBR322 plasmid and is therefore phase-locked 'on' such that all cells express type 1 fimbriae. E. coli F-18(pPR633) contains essentially the same plasmid minus the fimB gene and in L-broth about 30% of the cells express type 1 fimbriae. When fed alone to streptomycin-treated mice, E. coli F-18(pPKL91) colonized the large intestine at about 10(7) cfu/g of feces. However, when simultaneously fed with E. coli F-18(pPR633) at either high (10(10) cfu), or low doses (10(4) cfu), E. coli F-18(pPKL91) was a poor colonizer dropping to a level of between 10(2) and 10(3) cfu/g of feces. When given enough time to establish the state of colonization (10 days), E. coli F-18(pPKL91) persisted in feces in high numbers despite subsequent challenge by E. coli F-18(pPR633). Moreover, although both E. coli F-18(pPR633) and E. coli F-18(pPKL91) grew equally well in cecal mucus in vitro, E. coli F-18(pPR633) traveled through a layer of cecal mucus in vitro much faster than E. coli F-18(pPKL91). Together, the data suggest that type 1 fimbriated cells are at a disadvantage in initiating the colonization state because they have difficulty entering the mucus layer of the intestine as rapidly as non-fimbriated cells. The data also point to the possible biological significance of type 1 fimbrial phase-variation in the mouse large intestine.
 
Article
A DNA fragment encoding an approximately 18 kDa protein from Brucella abortus strain 2308 was cloned and expressed in Escherichia coli. This recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans. Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an approximately 8.7 kbp Sau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an approximately 3.0 kbp Pst 1 DNA fragment. DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17529. Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera. These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) from B. abortus strain 544 designated Omp 19.
 
Article
Previous results have demonstrated an essential role of gamma interferon (IFN-gamma) in resistance against Yersinia enterocolitica. Hence, we investigated the course of Yersinia infection in mice deficient for the IFN-gamma-inducing cytokines interleukin-12 (IL-12 p40(-/-)) or interleukin-18 (IL-18(-/-)). The experiments described herein argue for a critical role of both cytokines in protective immune responses against this pathogen.
 
Article
Identification of mycobacterial adhesins is needed to understand better the pathogenesis of tuberculosis and to develop new strategies to fight this infection. In this work, THP-1 monocytic cells were incubated with Mycobacterium tuberculosis culture filtrate proteins labelled with biotin and a dominant 19-kDa adhesin was found. This adhesin was characterized as the glycosylated and acylated 19-kDa antigen (Rv 3763). These findings were confirmed in assays with culture filtrate proteins and cell-wall fractions from a recombinant Mycobacterium smegmatis strain that overexpresses the 19-kDa antigen. Further, fluorescent microspheres coated with recombinant culture filtrate proteins adhere to cells in higher numbers than microspheres coated with native M. smegmatis proteins. The binding of the 19-kDa antigen to cells was inhibited with mannose receptor competitor sugars, Ca(2+) chelators and with a monoclonal antibody to the human mannose receptor. Phagocytosis assays showed high-level binding of bacilli to THP-1 cells that was inhibited with alpha-methyl-mannoside, mannan, EDTA and mAbs to the mannose receptor and to the 19-kDa M. tuberculosis antigen. Immunoprecipitation, cell-surface ELISA and immunostaining confirmed the expression of the mannose receptor by THP-1 cells. In conclusion, here we show that the macrophage mannose receptor, considered a pathogen pattern recognition receptor, may interact with mannose residues of mycobacterial glycoproteins that could promote the phagocytosis of mycobacteria.
 
Article
Pneumococcal surface protein A (PspA) is a virulence factor of Streptococcus pneumoniae that can elicit a protective antibody response. The pspA gene of strain Rx1 encodes a 65 kDa molecule composed of 588 amino acids. The N-terminal 288 amino acids are highly charged, and predict an alpha-helical coiled-coil protein structure. All monoclonal antibodies (MAbs) to PspA, obtained by screening against whole pneumococci, bind to the alpha-helical region of PspA, suggesting that this region is surface exposed. The C-terminal 217 amino acids of PspA contain the surface anchor of PspA and does not appear to be alpha-helical. In the middle of the molecule is a proline-rich region that is thought to traverse the cell wall. In this study we have mapped the immunogenic epitopes detected by 9 MAbs that were made against strain Rx1 PspA. Five of the MAb also react with the PspA of mouse virulent strain WU2. All epitopes were found in one of two portions of the alpha-helical region. One comprised the first 115 amino acids, and the other was within amino acids 192 and 260. The five MAbs that recognize WU2 PspA, but not the remaining four MAbs, were protective against strain WU2. The epitopes detected by four of the five protective MAbs mapped to region 192 to 260 of Rx1 PspA. The existence of protective epitopes in this region was confirmed by demonstrating that mice immunized with the cloned fragment containing these residues were protected from fatal infection with WU2. Since amino acids 192 to 260 are in the region of PspA anticipated to be adjacent to the cell wall, and probably well covered by capsule, the means by which antibodies to the region lead to protection is not obvious.
 
Article
One hundred seventy human uropathogenic Escherichia coli (UPEC) clinical isolates were compared with 35 E. coli strains isolated from feces of a control group to determine the presence of the set1, sen and astA genes encoding the ShET-1, ShET-2, and EAST toxins, respectively. Overall, 27 (16%), 8 (8%) and 0 UPEC isolates presented the set1B, the astA, and the sen genes, respectively. This is the first time the set gene has been found in UPEC clinical isolates.
 
Article
Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.
 
Article
Early growth of Salmonella typhimurium in spleen and liver of mice is controlled by the mouse chromosome 1 locus Ity/Nramp 1. Genetic control of resistance to the attenuated vaccine strain Rv6 of Salmonella abortusovis was studied in mice infected by the intravenous route. Comparison of kinetics of bacterial colonization of spleen and liver in two congenic BALB/c-susceptible (Itys) and -resistant (Ityr) mouse lines showed that BALB/c mice (Itys) were significantly more susceptible to infection than C.D2 mice (Ityr) suggesting that infection by this vaccine strain is controlled by a gene which is close or identical to Ity/Nramp 1. Congenic mice also differed in their anti-Salmonella antibody response, measured by ELISA: susceptible mice had a significantly higher antibody level than resistant mice, whatever the immunoglobulin isotype (IgM, IgG1, IgG2a, IgG3, IgA, and total immunoglobulins). The two congenic BALB/c mouse lines had equal serum C3c levels in response to infection. However, we observed a highly significant difference according to the sex of mice, suggesting a role of sex hormones in the regulation of the level of some complement factors. These results, obtained with congenic mice, strongly suggest that the Ity/Nramp 1 locus controls susceptibility to infection by the S. abortusovis vaccine strain Rv6 and influences the antibody response.
 
Article
Interleukin-1 (IL-1) mediates a number of proinflammatory biological responses that are thought to contribute to antibacterial resistance. In the present study we examined the ability of IL-1 to recruit inflammatory neutrophils and mononuclear phagocytes in vivo; a function that has been reported to be closely related to antibacterial resistance. Intraperitoneal injection of small amounts (1-10 LAF units) of purified human or recombinant murine IL-1 alpha (rIL-alpha) resulted in an increased influx of inflammatory neutrophils into the peritoneal cavity that peaked at 4-14 h after IL-1 injection. A small but consistent increase in peritoneal macrophages also was observed at 72 h after rIL-1 alpha injection. The ability of rIL-1 alpha to induce neutrophil accumulation was uninfluenced by polymyxin B, was sensitive to heat treatment (100 degrees C for 1 h), and was observed after i.p. injection into LPS-nonresponsive C3H/HeJ mice. These three lines of evidence suggested that contaminating LPS did not contribute substantially to rIL-1 alpha induced accumulation of neutrophils. Treatment of mice with indomethacin or nordihydroguaiaretic acid did not abrogate rIL-1 alpha induced neutrophil accumulation. Mice injected i.p. with increasing amounts of rIL-1 alpha demonstrated a corresponding enhancement of their resistance to an i.p. L. monocytogenes challenge 4 h later, thus suggesting that IL-1 mediated inflammatory phagocyte accumulation may contribute in part to nonspecific antibacterial resistance.
 
Article
We have previously demonstrated that administration of recombinant rIL-1 alpha enhances resistance against Listeria monocytogenes infection in mice. In this study we considered the possibility that this cytokine might also augment adoptive immunity conferred by the transfer of listeria-immune spleen cells. Concomitant administration of rIL-1 alpha with large numbers (2 x 10(7) or 10(8)) of listeria-immune spleen cells reduced the protection mediated by the transferred cells. Conversely, rIL-1 alpha co-administered with suboptimal numbers (1-5 x 10(6)) of immune splenocytes augmented anti-listeria resistance in an additive fashion. Although transfer of 10(6) listeria-immune spleen cells alone did not result in significant protection, when 10(6) immune cells were incubated with rIL-1 alpha prior to transfer they conferred significant protection to naive recipients. Time course experiments indicated that the greatest protection was achieved when listeria-immune spleen cells were pretreated with rIL-1 alpha for 2 h prior to adoptive transfer. The protection transferred by 10(6) rIL-1 alpha-pretreated immune spleen cells was not inhibited by TGF beta. This study is the first to use rIL-1 alpha to potentiate the adoptive transfer of resistance to an infectious agent by immune cells.
 
Top-cited authors
Didier Raoult
  • Aix-Marseille Université
Emmanouil Angelakis
  • URMITE CNRS-IRD 198 UMR 6236
Mical Paul
  • Rambam Medical Center
Azra Kamili
  • University of Kashmir
Matthieu Million
  • Aix-Marseille Université