Michel Frenette

Functional domains of colicin A

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor‐binding properties of each protein were also analysed. From these results, we suggest that the NH2‐terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor‐binding domain. The COOH‐terminal domain (residues 389 to 592) carries the pore‐forming activity.

Candida albicans and Streptococcus salivarius modulate IL-6, IL-8, and TNF-alpha expression and secretion by engineered human oral mucosa cells.

We investigated the involvement of oral epithelial cells via two cytokines (IL‐6 and TNF‐α) and one chemokine (IL‐8) in local defences against live yeast (Candida albicans) and bacteria (Streptococcus salivarius) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL‐6, IL‐8 and TNF‐α mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL‐6 levels were higher after contact with C. albicans than with S. salivarius. IL‐8 levels after contact with S. salivarius were higher than with C. albicans. Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans. These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria.

The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

BackgroundStreptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2) are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor.ResultsTwo mutants (G6G and M3G) possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757) that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9%) and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%), which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200), motif II (His239), and motif III (Ser568). SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly) at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p < 0.001).ConclusionIn summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

ABSTRACT In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABMan as well as membrane fragments containing IICMan and IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.

An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system.

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida.

Characterization of a galactokinase-positive recombinant strain of Streptococcus thermophilus.

ABSTRACT The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal− strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal+ strain grew and produced acid more rapidly than the Gal− wild-type strain.

Ribosomal DNA polymorphisms in the yeast Geotrichum candidum

The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.

Antimicrobial activity of nisin against the swine pathogen Streptococcus suis and its synergistic interaction with antibiotics

Streptococcus suis serotype 2 is known to cause severe infections in pigs, including meningitis, endocarditis and pneumonia. Furthermore, this bacterium is considered an emerging zoonotic agent. Recently, increased antibiotic resistance in S. suis has been reported worldwide. The objective of this study was to evaluate the potential of nisin, a bacteriocin of the lantibiotic class, as an antibacterial agent against the pathogen S. suis serotype 2. In addition, the synergistic activity of nisin in combination with conventional antibiotics was assessed. Using a plate assay, the nisin-producing strain Lactococcus lactis ATCC 11454 proved to be capable of inhibiting the growth of S. suis (n=18) belonging to either sequence type (ST)1, ST25, or ST28. In a microdilution broth assay, the minimum inhibitory concentration (MIC) of purified nisin ranged between 1.25 and 5 μg/mL while the minimum bactericidal concentration (MBC) was between 5 and 10 μg/mL toward S. suis. The use of a capsule-deficient mutant of S. suis indicated that the presence of this polysaccharidic structure has no marked impact on susceptibility to nisin. Following treatment of S. suis with nisin, transmission electron microscopy observations revealed lysis of bacteria resulting from breakdown of the cell membrane. A time-killing curve showed a rapid bactericidal activity of nisin. Lastly, synergistic effects of nisin were observed in combination with several antibiotics, including penicillin, amoxicillin, tetracycline, streptomycin and ceftiofur. This study brought clear evidence supporting the potential of nisin for the prevention and treatment of S. suis infections in pigs.

Detection of Variants of the pRAS3, pAB5S9, and pSN254 Plasmids in Aeromonas salmonicida subsp. salmonicida: Multidrug Resistance, Interspecies Exchanges, and Plasmid Reshaping

ABSTRACT The ubiquitous water-borne Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a worldwide disease in fish farms. Plasmids carrying antibiotic resistance genes have already been described for this bacterium. The aim of the present study was to identify and characterize additional multidrug resistance plasmids in A. salmonicida subsp. salmonicida. We sequenced the plasmids present in two multiple antibiotic-resistant isolates using high-throughput technologies. We also investigated 19 other isolates with various multidrug resistance profiles by genotyping PCR and assessed their resistance to tetracycline. We identified variants of the pAB5S9 and pSN254 plasmids that carry several antibiotic resistance genes and that have been previously reported in bacteria other than A. salmonicida subsp. salmonicida, which suggests a high level of interspecies exchange. Genotyping analyses and the antibiotic resistance profiles of the 19 other isolates support the idea that multiple versions of pAB5S9 and pSN254 exist in A. salmonicida subsp. salmonicida. We also identified variants of the pRAS3 plasmid. The present study revealed that A. salmonicida subsp. salmonicida harbors a wide variety of plasmids, which suggests that this ubiquitous bacterium may contribute to the spread of antibiotic resistance genes in the environment.

Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100 degrees C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 x 10(6) to 40 x 10(6) Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 microm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/phi, where X represents a modified amino acid residue and phi represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus.