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Geotrichum candidum is an ubiquitous yeast, and an essential component in the production of many soft cheeses. We developed a Multi-Locus Sequence Typing (MLST) scheme with five retained loci (NUP116, URA1, URA3, SAPT4, PLB3) which were sufficiently divergent to distinguish 40 Sequence Types (STs) among the 67 G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a PCR inter-LTR scheme, a fast and reproducible RAPD-like method for G. candidum, to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analyzed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species.

Kazachstania saulgeensis is a recently described species isolated from French organic sourdough. Here, we report the high quality genome sequence of a monosporic segregant of the type strain of this species, CLIB 1764(T) (= CBS 14374(T)). The genome has a total length of 12.9 Mb and contains 5326 putative protein-coding genes, excluding pseudogenes and transposons. The nucleotide sequences were deposited into the European Nucleotide Archive under the genome assembly accession numbers FXLY01000001-FXLY01000017.

WOS:000393357900048; International audience; Five ascosporogenous yeast strains related to the genus Kazachstania were isolated. Two strains (CLIB 1764(T) and CLIB 1780) were isolated from French sourdoughs; three others (UFMG-CM-Y273(T), UFMG-CM-Y451 and UFMG-CM-Y452) were from rotting wood in Brazil. The sequences of the French and Brazilian strains differed by one and three substitutions, respectively, in the D1/D2 large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS). The D1/D2 LSU rRNA sequence of these strains differed by 0.5 and 0.7 % from Kazachstania exigua, but their ITS sequences diverged by 8.1 and 8.3%, respectively, from that of the closest described species Kazachstania barnettii. Analysis of protein coding sequences of RPB1, RPB2 and EF-1 alpha distinguished the French from the Brazilian strains, with respectively 3.3, 6 and 11.7 % substitutions. Two novel species are described to accommodate these newly isolated strains: Kazachstania saulgeensis sp. nov. (type strain CLIB 1764(T)=CBS 14374(T)) and Kazachstania serrabonitensis sp. nov. (type strain UFMG-CM-Y273(T)=CLIB 1783(T)=CBS 14236(T)). Further analysis of culture collections revealed a strain previously assigned to the K. exigua species, but having 3.8% difference (22 substitutions and 2 indels) in its ITS with respect to K. exigua. Hence, we describe a new taxon, Kazachstania australis sp. nov. (type strain CLIB 162(T)=CBS 2141(T)), to accommodate this strain. Finally, Candida humilis and Candida pseudohumilis are reassigned to the genus Kazachstania as new combinations. On the basis of sequence analysis, we also propose that Candida miller and Kazachstania humilis comb. nov. are conspecific.

The neisserial Lip antigen is a conserved antigen associated with the pathogenic Neisseria species, and is composed of multiple repeats of a consensus pentapeptide. A series of monoclonal antibodies reacting with meningococcal Lip antigen were subjected to epitope mapping, using solid-phase synthetic peptides based on the consensus repeat sequence. The antibodies were found to recognize different continuous epitopes based on the consensus sequence. One monoclonal antibody was utilized in affinity chromatography to obtain purified Lip antigen and the antigen was used for immunization of mice. The resulting antisera did not recognize Lip antigen on Western blots but reacted specifically with Lip antigen in immune precipitation experiments, indicating that the predominant polyclonal immune response was directed against conformational epitopes. Despite the diversity of both continuous and conformational epitopes recognized by the antibodies produced, none of the antibodies demonstrated the ability to promote complement-mediated bactericidal activity. Thus despite its initial apparent promise as a potential vaccine candidate the case for the inclusion of Lip antigen in vaccine formulation cannot be supported at present.

Monoclonal antibody SM24 recognizes a protective, highly conserved but non-immunogenic epitope on outer membrane protein PIB of Neisseria gonorrhoeae . A series of overlapping synthetic peptides, spanning the deduced amino acid sequence of PIB from strain R10, have been synthesized on solid phase supports. Monoclonal antibody SM24 reacted with two adjacent decapeptides corresponding to residues 191–200 and 196–205, containing the common sequence TYSIP. Following localization of the epitope recognized, a peptide was synthesized corresponding to residues 193–204. The peptide was coupled to a carrier protein (KLH) and both the free peptide and peptide-KLH conjugate were used for immunization of rabbits. The resulting antisera reacted with the immunizing peptide, with denatured PIB on Western blots and, in addition, with native PIB in outer membranes of both the homologous and a heterologous strain. In the presence of human complement the sera were bactericidal for both the homologous and the heterologous strain. Thus synthetic peptides may be used to induce a protective polyclonal immune response against epitopes on gonococcal PI which are normally only weakly or non-immunogenic.

The type IV pili (Tfp) of Neisseria meningitidis play an essential role in meningococcal virulence by mediating the initial interaction of bacteria with host cells. Tfp are also subject to retraction, which relies on the PilT protein. Among the other components of the Tfp machinery, PilC1, a pilus-associated protein, is important for Tfp biogenesis and adhesion. Adhesion of N. meningitidis to living epithelial cells was previously shown to rely on the upregulation of the pilC1 gene. On the other hand the lack of induction of pilC1 is believed to be responsible for the low adhesion of N. meningitidis onto fixed dead cells. Surprisingly, a pilT mutant, unable to retract its pili, has been shown to adhere very efficiently onto both living and fixed epithelial cells. To elucidate the mechanisms by which the pilus retraction machinery mediates meningococcal adhesion onto fixed cells, an analysis of gene expression levels in wild-type and pilT meningococci was performed using DNA microarrays. One of the upregulated genes in the pilT strain was pilC1. This result was confirmed using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis. The transcription starting point responsible for the upregulation of pilC1 in a pilT background was shown to be different from those controlling the induction of pilC1 upon contact with living host cells. Subsequent work using a strain hyperproducing PilT confirmed that PilT downregulates the production of PilC1. Furthermore using a pilC1 allele under the control of IPTG, we demonstrated that the upregulation of pilC1 in a pilT background was responsible for the adhesive phenotype onto fixed dead cells. Taken together our results demonstrate that the pilus retraction machinery negatively controlled the adhesiveness of the Tfp via the expression of pilC1.

International audience; An increasing number of pathogenicity factors are discovered to be carried by bacteriophages. This review considers the bacteriophage-bacterium interaction and its relation to disease processes. We discuss the search for new bacteriophage-associated pathogenicity factors with emphasis on recent advances brought by the use of genomic sequence data and the techniques of genomic epidemiology.

Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109-11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 degrees C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a functional periplasmic DsbA.

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The class 4 protein of Neisseria meningitidisis is a highly conserved outer membrane protein, closely related to the protein PIII of Neisseria gonorrhoea. Monoclonal antibodies SM50 and SM54 raised against PIII also react with class 4 protein but do not promote complement mediated bactericidal killing of meningococci. In addition, mAb SM50 inhibits the antimeningococcal bactericidal activity both of normal human sera and of mAb SM300, directed against a protective epitope on the class 1 outer membrane protein. The ability of class 4 protein to induce such ‘blocking’ antibodies suggests that its presence in experimental vaccines, based on meningococcal outer membranes, may be antagonistic to the development of effective bactericidal immunity.