J. Daniel Dubreuil

Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity

Abstract Actinobacillus pleuropneumoniae is an important pig pathogen that is responsible for swine pleuropneumonia, a highly contagious respiratory infection. Knowledge of the importance, composition and structural determination of the major antigens involved in virulence provides crucial information that could lead to the development of a rationale for the production of specific serodiagnostic tools as well as vaccine development. Thus, efforts have been devoted to study mainly A. pleuropneumoniaevirulence determinants with special emphasis on the Apx toxins (for A. pleuropneumoniaeRTX toxins). In comparison, little attention has been given to the surface polysaccharides, which include capsular polysaccharides (CPS) and cell-wall lipopolysaccharides (LPS). Here, we review current knowledge on CPS and LPS of A. pleuropneumoniae used as diagnostic tools to monitor the infection and as immunogens for inclusion in vaccine preparations for animal protection.

Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 (EAST1): A New Toxin with an Old Twist

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) is a small protein that was first detected more than a decade ago in an enteroaggregative E. coli (EAEC) strain isolated from the stools of a diarrheic child. The EAST1 gene, astA, is not solely present in EAEC, but also in other categories of diarrheagenic E. coli. Strains expressing EAST1 have been shown to induce diarrhea principally in humans, although they have also been associated with piglets and calves. EAST1 toxin has been proposed as a virulence factor implicated in the mechanism of pathogenesis of EAEC and could play a role in the pathogenicity of other enteropathogens as well. This toxin is often compared to E. coli STa enterotoxin because they share some physical and mechanistic similarities. This review summarizes the various observations on EAST1 since its discovery.

Identification of a Surface Protein of Streptococcus suis and Evaluation of Its Immunogenic and Protective Capacity in Pigs

ABSTRACT A Streptococcus suis surface protein reacting with convalescent-phase sera from pigs clinically infected by S. suis type 2 was identified. The apparent 110-kDa protein, designated Sao, exhibits typical features of membrane-anchored surface proteins of gram-positive bacteria, such as a signal sequence and an LPVTG membrane anchor motif. In spite of high identity with the partially sequenced genomes of S. suis Canadian strain 89/1591 and European strain P1/7, Sao does not share significant homology with other known sequences. However, a conserved avirulence domain that is often found in plant pathogens has been detected. Electron microscopy using an Sao-specific antiserum has confirmed the surface location of the Sao protein on S. suis. The Sao-specific antibody reacts with cell lysates of 28 of 33 S. suis serotypes and 25 of 26 serotype 2 isolates in immunoblots, suggesting its high conservation in S. suis species. The immunization of piglets with recombinant Sao elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.

Inactivation of the Pst System Reduces the Virulence of an Avian Pathogenic Escherichia coli O78 Strain

ABSTRACT Escherichia coli O78 strains are frequently associated with extraintestinal diseases, such as airsacculitis and septicemia, in poultry, livestock, and humans. To understand the influence of the pst operon in the virulence of E. coli, we introduced mutations into the pst genes of the avian pathogenic E. coli (APEC) O78:K80 strain χ7122 by allelic exchange. The mutation of pst genes led to the constitutive expression of the Pho regulon. Furthermore, the virulence of APEC strain χ7122 in a chicken infection model was attenuated by inactivation of the Pst system. The pst mutant caused significantly fewer extraintestinal lesions in infected chickens, and bacterial numbers isolated from different tissues after infection were significantly lower for the mutant than for the wild-type strain. Moreover, resistance to the bactericidal effects of rabbit serum and acid shock was impaired in the pst mutant, in contrast to the wild-type strain. In addition, the MIC of polymyxin was twofold lower for the mutant than for the wild-type strain. Although the pst mutant demonstrated an increased susceptibility to rabbit serum, this strain was not killed by chicken serum, suggesting the presence of differences in host innate immune defenses and complement-mediated killing. In APEC O78 strain χ7122, a functional Pst system is required for full virulence and resistance to acid shock and polymyxin. Our results suggest that the mutation of pst genes induces a deregulation of phosphate sensing and changes in the cell surface composition that lead to decreased virulence, indicating the importance of the Pst system for the virulence of pathogenic E. coli strains from different hosts.

Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella

The distribution and expression of the astA gene (EAST1 toxin) among 358 strains of Enterobacteriaceae were studied. The gene was found in 32.6% and 11.9% of Escherichia coli and Salmonella strains, respectively. The majority of E. coli EAST1-positive strains were found among EHEC (88.0%), EAggEC (86.6%), and A-EPEC (58.3%). The gene was present in 16.6% of E. coli strains without known virulence genes. There was no significant variation among the different serotypes of E. coli tested regarding the presence of the gene. For EPEC, 13.7% of the tested strains were astA-positive. Among atypical EPEC (eae+, bfp-, EAF-) and (eae+, bfp+, EAF-) 46.2 and 72.7%, respectively, were positive. The majority of the A-EPEC (87%) and EaggEC (83%) strains expressed the EAST-1 toxin as judged from Ussing chamber experiments. Of 32 EIEC strains studied, 2 possessed and expressed the gene as determined in Ussing chamber experiments. Among the Salmonella strains studied, five strains isolated from food were positive for astA and one strain of S. agona showed biological activity in Ussing chamber experiments.

Role of Heat-Stable Enterotoxins in the Induction of Early Immune Responses in Piglets after Infection with Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat - labile (LT) enterotoxins are cause of post – weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac+, LT+ STa+ STb+ ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

Pasteurella multocida toxin stimulates mitogenesis and cytoskeleton reorganization in Swiss 3T3 fibroblasts

Pasteurella multocida toxin (PMT) causes cytoplasmic retraction in epithelial cells, activates osteoclast neoformation, and is a potent mitogen for Swiss 3T3 fibroblasts. In the present study designed to further investigate the effects of PMT on cell shape and proliferation, we report that the mitogenic effect of affinity‐purified PMT on quiescent 3T3 cells was even superior at 5 ng/ml to that of fetal bovine serum or bombesin. This positive effect was inhibited by heat denaturation and methylamine treatment (this agent blocks internalization). Preincubation of PMT with gangliosides GM1, GM2, or GM3 counteracted its effect on DNA synthesis, suggesting that the toxin binds to GM‐type ceramides on target cells. The distribution of F‐actin was analyzed in control/treated cells using FITC‐conjugated phalloidin. In comparison with FBS and bombesin, PMT triggered a more rapid and profound reorganization of cortical actin into prominent stress fibers after only 5–10 min. This event lead to the retraction of cells after only 30 min and ultimately to the induction of mitotic figures. Interestingly, methylamine blocked the effects of PMT on stress fiber formation and cell retraction but not the ruffling response, suggesting that some early events may not require toxin internalization. In summary, these findings indicate that PMT concomitantly exerts a strong mitogenic activity and a rapid stimulation of cytoskeletal rearrangements, possibly after binding to membrane gangliosides and subsequent internalization. We propose that this toxin could be used in the future as a defined inducer of transduction signals involved in cellular proliferation and control of cell shape.

Evaluation of long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 for the serodiagnosis of swine pleuropneumonia

Long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 have been evaluated and compared with a crude boiled extract (CBE) in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. The mean optical density (OD) obtained with the LC-LPS in ELISA using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotype 5 was not significantly different from that obtained with the CBE. However, sera from animals exposed to serotypes of A. pleuropneumoniae other than serotype 5 presented a significantly lower mean OD (P < 0.05) when the LC-LPS was used. As a consequence, it was demonstrated that a high percentage of non-specific cross-reactions were eliminated, without losing specificity. The specificity and the sensitivity of the LC-LPS- and CBE-ELISA were evaluated using two different cut-off values (the OD plus two and three standard deviations) (SD) obtained from 593 sera from negative herds. The LC-LPS appeared a more suitable antigen than the CBE, since the sensitivity and the specificity (obtained with both thresholds) were statistically improved (P < 0.01). A threshold of 0.244 (mean OD plus three SD) for the LC-LPS-ELISA seemed more suitable, since a sensitivity of 79% and a specificity of 97% was achieved. Nevertheless, it may be advisable to keep a buffer range (OD between 0.194 and 0.243) and to consider sera presenting values within this range as suspicious. In the present study, the complement fixation test presented a high specificity (97%) and a very low sensitivity (47%). A herd with animals presenting ELISA positive and CFT negative results in serology, along with the absence of suggestive lesions should not be considered as a non-infected herd.

Escherichia coli Heat-Stable Toxin b Impairs Intestinal Epithelial Barrier Function by Altering Tight Junction Proteins

ABSTRACT Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.

Modulation of Hexa-Acyl Pyrophosphate Lipid A Population under Escherichia coli Phosphate (Pho) Regulon Activation

Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon. We showed that a pst mutation results in virulence attenuation in extraintestinal pathogenic Escherichia coli (ExPEC) strains. Our results indicate that the bacterial cell surface of the pst mutants is altered. In this study, we show that pst mutants of ExPEC strains display an increased sensitivity to different cationic antimicrobial peptides and vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is significantly less abundant in pst mutants. Among differentially expressed genes in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to lipid A, forming the 1-diphosphate species, was found to be downregulated. Our results strongly suggest that the Pho regulon is involved in lipid A modifications, which could contribute to bacterial surface perturbations. Since the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A modification mechanism could be widely distributed among gram-negative bacterial species.