Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans.

The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 proteins secreted during biofilm growth. Further to confirming the release of established virulence factors (e.g. cytolethal distending toxin [CDT], and leukotoxin [LtxA]), we identified additional putative virulence determinants in the secretome. These included DegQ, fHbp, LppC, Macrophage infectivity protein (MIP), NlpB, Pcp, PotD, TolB, and TolC. This finding indicates that the number of extracellular virulence-related proteins is much larger than previously demonstrated, which was also supported by in silico analysis of the strain D7S genome. Moreover, our LC-MS/MS and in silico data revealed that at least Type I, II, and V secretion are actively used to excrete proteins directly into the extracellular space, or via two-step pathways involving the Sec/Tat systems for transport across the inner membrane, and outer membrane factors, secretins and auto-transporters, respectively for delivery across the outer membrane. Taken together, our results provide a molecular basis for further elucidating the role of A. actinomycetemcomitans in periodontal and systemic diseases.

MALDI-TOF mass spectrometry confirms clonal lineages of Gallibacterium anatis between chicken flocks.

Gallibacterium anatis has been suggested to have a causal role in the salpingitis/peritonitis complex in chickens, beside its isolation from the respiratory tract. As G. anatis strains from different flocks were compared by MALDI-TOF MS proteomic phenotyping it could be demonstrated that in most flocks one clonal lineage was present. This finding is also reflected by data achieved when isolates from different organs within a bird generally belong to the same clonal lineage. In addition, it was also confirmed by two independent experiments, as well as, two MALDI instruments. Altogether, proteomic phenotyping indicates that the nature of a chicken flock may play a certain role in particular clone type selection of G. anatis.

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge.

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.

Phocoenobacter uteri gen. nov., sp. nov., a new member of the family Pasteurellaceae Pohl (1979) 1981 isolated from a harbour porpoise (Phocoena phocoena).

Phenotypic and phylogenetic studies were performed on a Gram-negative, rod-shaped bacterium isolated from the uterus of a porpoise. Biochemical and physiological studies indicated that the bacterium was related to the family Pasteurellaceae. Comparative 16S rRNA gene sequencing studies confirmed these findings and demonstrated that the bacterium represents a hitherto unknown subline within this family of organisms. Based on the results of the phylogenetic analysis and phenotypic criteria, it is proposed that the bacterium be assigned to a new genus, Phocoenobacter uteri gen. nov., sp. nov. The type strain of Phocoenobacter uteri sp. nov. is NCTC 12872T.