Identification, cloning and characterization of SpEX exotoxin produced by Staphylococcus pseudintermedius

Staphylococci have evolved numerous strategies to evade their hosts’ immune systems. Some staphylococcal toxins target essential components of host innate immunity, one of the two main branches of the immune system. Analysis of the Staphylococcus pseudintermedius secretome using liquid chromatography mass spectrometry guided by genomic data, was used to identify an S. pseudintermedius exotoxin provisionally named SpEX. This exoprotein has low overall amino acid identity with the Staphylococcus aureus group of proteins named staphylococcal superantigen like proteins (SSLs) and staphylococcal enterotoxin- like toxin X (SEIX), but predictive modeling showed that it shares similar folds and domain architecture to these important virulence factors. In this study, we found SpEX binds to complement component C5, prevents complement mediated lysis of sensitized bovine red blood cells, kills polymorphonuclear leukocytes and monocytes and inhibits neutrophil migration at sub-lethal concentrations. A mutant version of SpEX, produced through amino acid substitution at selected positions, had diminished cytotoxicity. Anti-SpEX produced in dogs reduced the inhibitory effect of native SpEX on canine neutrophil migration and protected immune cells from the toxic effects of the native recombinant protein. These results suggest that SpEX likely plays an important role in S. pseudintermedius virulence and that attenuated SpEX may be an important candidate for inclusion in a vaccine against S. pseudintermedius infections

Complete Genome Sequence of Staphylococcus pseudintermedius Type Strain LMG 22219

We report the first complete genome sequence of LMG 22219 (=ON 86T = CCUG 49543T), the Staphylococcus pseudintermedius type strain isolated from feline lung tissue. This sequence information will facilitate phylogenetic comparisons of staphylococcal species and other bacteria at the genome level

Characterization of a leukocidin identified in Staphylococcus pseudintermedius

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermediussecretome using liquid chromatography mass spectrometry we identified two proteins, named “LukS-I” and “LukF-I”, encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermediusLukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermediusLukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections

Staphylococcus pseudintermedius Sbi paralogs inhibit complement and bind IgM, IgG Fc and Fab

The success of staphylococci as pathogens has been attributed, in part, to their ability to evade their hosts’ immune systems. Although the proteins involved in evasion have been extensively studied in staphylococci affecting humans little characterization has been done with Staphylococcus pseudintermedius, an important cause of pyoderma in dogs. Staphylococcus aureus binder of immunoglobulin (Sbi) interferes with innate immune recognition by interacting with multiple host proteins. In this study, a S. pseudintermedius gene that shares 38% similarity to S. aureus Sbi was cloned from S. pseudintermedius strains representative of major clonal lineages bearing two paralogs of the protein. Binding of immunoglobulins and Fab and Fc fragments as well as interaction with complement was measured. S. pseudintermedius Sbi protein bound IgG from multiple species and canine complement C3, neutralized complement activity and bound to canine IgM and B cells. Evidence from this work suggests Sbi may play an important role in S. pseudintermedius immune evasion

Complete Genome Sequences of Three Staphylococcus pseudintermedius Strains Isolated from Botswana

We report here the first whole-genome sequences for 3 strains of Staphylococcus pseudintermedius (112N, 113N, and 114N) isolated in Africa. Samples of this opportunistic pathogen were collected from nasal swabs obtained from healthy carrier dogs in Botswana. The sequence information will facilitate spatial phylogenetic comparisons of staphylococcal species and other bacteria at the genome level

Isolation of Bartonella sp. from Sheep Blood

A Bartonella sp. was isolated from sheep blood. Bacterial identification was conducted by using electron microscopy and DNA sequencing of the 16S rRNA, citrate synthase, riboflavin synthase, and RNAase P genes. To our knowledge, this is the first report of ovine Bartonella infection

Complete Genome Sequences of Four Staphylococcus aureus Sequence Type 398 Isolates from Four Goats with Osteomyelitis

Staphylococcus aureus is the causative agent of multiple infections, including bacteremia, infective endocarditis, osteomyelitis, septic arthritis, and prosthetic device infections. We report here the first whole-genome sequence for four S. aureus sequence type 398 isolates from clinical cases of osteomyelitis in four goats with a history of orthopedic surgery

Complete Genome Sequences of Three Important Methicillin-Resistant Clinical Isolates of Staphylococcus pseudintermedius.

We report the first complete genome sequences of three predominant clones (ST68, ST71, and ST84) of methicillin-resistant Staphylococcus pseudintermedius in North America. All strains were isolated from canine infections and have different SCCmec elements and antibiotic resistance gene patterns

Epidemiological observations on pastern dermatitis in young horses and evaluation of essential fatty acid spot-on applications with or without phytosphingosine as prophylactic treatment

Background - Equine pastern dermatitis (EPD) is a common multifactorial clinical syndrome in horses. Treatment can be difficult;pathogenesis and triggering factors cannot always be determined. Objectives - To assess risk factors for developing EPD in a large group of horses kept under the same conditions and to analyse whether or not a spot-on containing essential fatty acids and antimicrobial agents is able to prevent the development of EPD or accelerate the healing process. Animals - Each year 50 young, privately owned, warmblood horses were prospectively included. Methods - All horses were examined weekly between August and October for the presence of typical EPD skin lesions. Additionally, in the first year, horses were randomly divided into three subgroups of intervention. The pastern areas were treated once weekly either with 0.6 mL of a spot-on containing essential fatty acids and aromatic oils, or a preparation containing additional antibacterial phytosphingosine, or not at all. Results - Nonpigmented pastern areas were affected significantly more often than pigmented pastern areas (P < 0.0001). The interaction between moisture and opportunistic pathogens seemed to be a major triggering factor for EPD. There was no difference in the occurrence of EPD in the three subgroups. The lesion scores of affected limbs in both spot-on groups were significantly lower compared to the control group. Conclusion and clinical importance - Moisture and lack of pigmentation predisposed to EPD. Topical application of the tested spot-on once weekly did not prevent the disease. A positive effect of both spot-on products on the severity of EPD lesions was detected

Inferring gene coexpression networks for low dose ionizing radiation using graph theoretical algorithms and systems genetics

Background Biological data generated through large scale -omics technologies have resulted in a new paradigm in the study of biological systems. Instead of focusing on individual genes or proteins these technologies enable us to extract biological networks using powerful computing and statistical algorithms that are scalable to very large datasets. Materials and methods We have developed a tool chain using novel graph algorithms to extract gene coexpression networks from microarray data. We highlight implementation of our tool chain to investigate the effects of in vivo low dose ionizing radiation treatments on mice. We are using systems genetics approach to investigate the biological effects of low dose (10 cGy) ionizing radiation. We measured the base line gene expression profile from spleen tissue of BXD recombinant inbred mice using Illumina microarrays. The data was filtered using coefficient of variance after robust spline normalization and variance stabilizing transformation. A graph was then derived from this data, with probes as vertices and edges between them representing correlations. The graph was analyzed using our toolkit to find the size and number of maximal cliques. We deployed another tool called paraclique that relaxes clique’s requirement that every edge be present between all vertices. Paraclique enables us to account for inherent noise in the microarray data and stochastic nature of biological processes. Using immunophenotype data from the baseline BXD mice, we employed biclique analysis to determine interactions between genotypes and immunophenotypes (%CD4, %CD3, LN T:B, %CD8, and LN CD4:CD8). We also extracted eQTLs from BXD data using QTL-Reaper from base line gene expression profiles. 1881 transcripts were associated with 686 loci. The eQTLs were classified as cis or trans according to their genomic positions. Besides population level studies we also investigated the differential effect of low dose and high dose (1Gy) of ionizing radiations on spleen gene expression in inbred parental strains (C57BL/6J and DBA/2J) of BXD recombinant inbred mice as well as BALB/c mice, a known radiation-sensitive strain